Antibodies to MASP-2

ABSTRACT

The invention relates to antibodies to MASP-2 and functional equivalents thereof. In particular, the invention relates to MASP-2 antibodies capable of inhibiting the function of MASP-2. The invention furthermore discloses MASP-2 epitopes, wherein antibodies recognising said epitopes are in particularly useful for inhibiting MASP-2 activity. The invention also relates to methods of producing said antibodies, methods of inhibiting MASP-2 activity as well as to pharmaceutical compositions comprising the MASP-2 antibodies.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of prior U.S. patent applicationSer. No. 14/745,247, filed Jun. 19, 2015, which is a divisional of priorU.S. patent application Ser. No. 10/556,509, filed Aug. 23, 2006, nowissued as U.S. Pat. No. 9,096,676, priority from the filing date ofwhich is hereby claimed under 35 U.S.C. § 120, which is a 371 NationalPhase Application of International PCT Application No. PCT/DK04/00338,filed May 12, 2004, now lapsed, which claims priority to Danish PatentApplication No. PA 2003 00716, filed May 12, 2003, now lapsed.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided intext format in lieu of a paper copy and is hereby incorporated byreference into the specification. The name of the text file containingthe sequence listing is MP_1_0117_US3_SequenceListingasFiled_20181204. Kthe text file is 86 KB, was created on Dec. 3, 2018; and is beingsubmitted via EFS-Web with the filing of the specification.

FIELD OF INVENTION

The present invention relates to antibodies to MASP-2 and functionalequivalents thereof. In particular, the invention relates to MASP-2antibodies capable of inhibiting the function of MASP-2. Furthermore,the invention relates to methods of producing said antibodies, methodsof inhibiting MASP-2 activity as well as to pharmaceutical compositionscomprising the MASP-2 antibodies.

BACKGROUND OF INVENTION

The complement system comprises a complex array of enzymes andnon-enzymatic proteins of importance to the function of the innate aswell as the adaptive immune defense¹. Until recently two modes ofactivation were known, the classical pathway initiated byantibody-antigen complexes and the alternative pathway initiated bycertain structures on microbial surfaces. A third, novelantibody-independent pathway of complement activation has beendescribed². This pathway is initiated when mannan-binding lectin (MBL,first described as mannan-binding protein³, MBP, see Ezekowitz, U.S.Pat. No. 5,270,199) binds to carbohydrates and is known as the MBLectinpathway.

MBL is structural related to the C1q subcomponent of component C1 ofcomplement, and it appears that MBL activates the complement system viaan associated serine protease termed MASP⁴ or p100⁵, which is similar tothe C1 r and C1 s components of the classical pathway. The newcomplement activation pathway is called the MBLectin pathway. Accordingto the mechanism postulated for this pathway, MBL binds to specificcarbohydrate structures found on the surface of a range ofmicroorganisms including bacteria, yeast, parasitic protozoa andviruses⁵, and its antimicrobial activity results from activation of theterminal, lytic complement pathway components' or promotingphagocytosis⁸.

MASPs (MBL-associated serine protease) are serine proteases similar instructure to C1 r and C1 s of the complement pathway. MASP-1 has ahistidine loop structure of the type found in trypsin and trypsin-likeserine proteases. MASP-1 has been found to be involved in complementactivation by MBL. A cDNA clone encoding

MASP-1 has been reported that encodes a putative leader peptide of 19amino acids followed by 680 amino acid residues predicted to form themature peptide.

MASP-2 (MBL-associated serine protease 2)²² is a serine protease alsosimilar in structure to C1 r and C1 s of the complement pathway. Likethese, and contrary to MASP1, it has no histidine loop structure of thetype found in trypsin and trypsin-like serine proteases. MASP-2 has beenfound to be involved in complement activation by MBL.

Antibodies to MASP-2 has been described in the prior art.

WO 02/06460 describes human MASP-2. The document furthermore describesantibodies to MASP-2 raised by immunising rabbits with the N-terminal 19amino acids of human MASP-2 or chickens with aa 505 to 523 and aa 538 to556 of human MASP-2.

SUMMARY OF INVENTION

Interestingly, the inventors of the present invention have recognisedthat inhibition of the MBLectin pathway may be desirable in thetreatment of a number of clinical conditions. However, specificinhibitors of the MBLectin pathway are not well characterised in theprior art and therefore an unmet need of specific inhibitors exists.

The present invention discloses that antibodies to the C-terminal partof MASP-2 are capable of inhibiting the activity of MASP-2 moreefficiently than antibodies to the N-terminal part of MASP-2. Theinvention furthermore discloses MASP-2 epitopes, wherein antibodiesrecognising said epitopes are in particularly useful for inhibiting theactivity of MASP-2. Preferred epitopes are describes herein below.

In one aspect the invention relates to an antibody or a functionalequivalent thereof specifically recognising and binding at least part ofan epitope recognised by one or more reference antibodies selected fromthe group consisting of

-   -   i the monoclonal antibody produced by the hybridoma cell line        deposited under the deposition number 03050904;    -   ii the monoclonal antibody produced by the hybridoma cell line        designated M0545YM029;    -   iii the monoclonal antibody produced by the hybridoma cell line        designated M0545YM035;    -   iv the monoclonal antibody produced by the hybridoma cell line        designated M0545YM046; and    -   v the monoclonal antibody produced by the hybridoma cell line        designated M0545YM048.

Accordingly, it is an objective of the present invention to provideantibodies or functional equivalents thereof specifically recognisingand binding an epitope within the C-terminal part of MASP-2 or afunctional homologue thereof.

It is furthermore an objective of the present invention to provideisolated polypeptides comprising a C-terminal fragment of MASP-2, saidpolypeptide being useful for raising antibodies to epitopes within theC-terminus of MASP-2. In particular, the isolated polypeptides from theC-terminal part of MASP-2 or a functional homologue thereof, may bepolypeptides comprising or consisting of:

-   -   i. the EGF, CUB2, CCP1, CCP2 and serine protease domains; or    -   ii. the CUB2, CCP1, CCP2 and serine protease domains; or    -   iii. the CCP1 domain; or    -   iv. the CCP2 domain; or        the CCP1, and CCP2 domain

It is furthermore an objective of the present invention to providemethods of producing an antibody inhibiting MASP-2 activity, byimmunising an animal, preferably a mammal, with isolated polypeptidescomprising a C-terminal fragment of MASP-2, said polypeptide beinguseful for raising antibodies to epitopes within the C-terminus ofMASP-2. In particular, the isolated polypeptides from the C-terminalpart of MASP-2 or a functional homologue thereof, may be polypeptidescomprising or consisting of:

-   -   v. the EGF, CUB2, CCP1, CCP2 and serine protease domains; or    -   vi. the CUB2, CCP1, CCP2 and serine protease domains; or    -   vii. the CCP1 domain and the serine protease domain; or    -   viii. the CCP2 domain and the serine protease domain; or    -   ix. the CCP1, CCP2 and the serine protease domains    -   x. the serine protease domain

It is a further objective of the present invention to provide methods ofproducing an antibody specifically recognising and binding an epitopewithin the C-terminal part of MASP-2 or a functional homologue thereof,comprising the step of administering to a mammal the C-terminal part ofMASP-2 or a fragment thereof or a functional homologue thereof.Antibodies produced according to the method are also disclosed by theinvention.

It is an even further objective of the invention to provide methods ofinhibiting the activity of MASP-2 comprising the steps of

-   -   1) Providing a composition comprising MASP-2;    -   2) Providing a MASP-2 antibody according to the invention;    -   3) Incubating said composition with said antibody, thereby        inhibiting MASP-2 activity

Inhibition of MASP-2 will lead to inhibition of complement activation,preferably to inhibition of the MBLectin pathway. Accordingly,antibodies inhibiting the activity of MASP-2 may be used to inhibitactivation of the MBLectin pathway and accordingly, said antibodies maybe useful for treatment of clinical conditions characterised by improperactivation of complement, preferably improper activation of the MBLectinpathway.

Hence, the invention also relates to methods of inhibiting the MBLectinpathway, preferably said methods involve the use of antibodies toMASP-2, capable of inhibiting the activity of MASP-2.

It is a still further objective of the present invention to providepharmaceutical compositions comprising MASP-2 antibodies or functionalequivalents thereof recognising an epitope within the C-terminal part ofMASP-2 together with pharmaceutically acceptable excipients.

It is yet another objective of the present invention to provide amedicament for treatment of a clinical condition comprising an antibodyor a functional equivalent thereof recognising an epitope within theC-terminal part of MASP-2 as an active ingredient.

It is also an objective of the present invention to provide methods oftreatment of a clinical condition comprising administering to anindividual in need thereof a therapeutically effective dosage of anantibody or a functional equivalent thereof recognising an epitopewithin the C-terminus of MASP-2.

It is furthermore an objective of the present invention to provide usesof antibodies or functional equivalents thereof recognising an epitopewithin the C-terminal part of MASP-2, for the preparation of amedicament for the treatment of a clinical condition in an individual inneed thereof.

DESCRIPTION OF DRAWINGS

FIG. 1 depicts a schematic representation of MASP-2 indicating theindividual domains.

FIG. 2 shows an alignment of the human MASP-1 (SEQ ID NO:15), MASP-2(SEQ ID NO:14, C1r (SEQ ID NO:16) and C1s (SEQ ID NO:17) sequencesindicating the presence of the individual domains in MASP-2. Amino acidsconserved in the four proteins are furthermore indicated by asterisk.

FIG. 3 depicts inhibition of C4 deposition in full serum by a MASP-2antibody.

FIG. 4 depicts inhibition of C4 deposition by different concentrationsof a MASP-2 antibody.

FIG. 5 shows a C4 deposition assay. The figure illustrates inhibition ofC4 deposition in human serum using different purified anti-MASP-2antibodies.

FIG. 6 shows an assembly of Western blots against MASP-2 in human serumusing 4 different antibodies. Human serum was loaded on each lane. Thefigure is assembled from four separate Western blots using theantibodies shown in the lanes above for detection.

FIG. 7 shows the results of a competitive ELISA for determination ofoverlapping epitopes.

FIG. 8 illustrates the nucleotide sequence (SEQ ID NO:18) encoding thevariable region of the light chain of the NimoAb101 antibody(DWE16140-6cons) (SEQ ID NO:19).

FIG. 9 illustrates the nucleotide sequence (SEQ ID NO:20) encoding thevariable region of the heavy chain of NimoAb101 antibody(DWE16140-3consRev) (SEQ ID NO:21).

FIG. 10 shows an alignment between sequences of the heavy chain of theNimoAb101 antibody (DWE16140-1con (SEQ ID NO:29), DWE16140-2con (SEQ IDNO:30), DWE16140-3con (SEQ ID NO:31), DWE16140-4con (SEQ ID NO:27),DWE16140-5con (SEQ ID NO:28), and DWE16140-8con (SEQ ID NO:32)) togetherwith homologous sequences P18525 (SEQ ID NO:22), P18526 (SEQ ID NO:23),P18529 (SEQ ID NO:24), P01764 (SEQ ID NO:25), P01783 (SEQ ID NO:26),P01868 (SEQ ID NO:33), P01869 (SEQ ID NO:34), P20759 (SEQ ID NO:35) andP20760 (SEQ ID NO:36) identified by BLAST searches.

FIG. 11 shows an alignment between sequences of the light chain of theNimoAb101 antibody (DWE16140-6con (SEQ ID NO:37), DWE16140-7con (SEQ IDNO:38), DWE16140-9con (SEQ ID NO:39) and DWE16140-10con (SEQ ID NO:40))together with homologous sequences P01594 (SEQ ID NO: 41), P01595 (SEQID NO: 42), P01635 (SEQ ID NO: 43), P01636 (SEQ ID NO: 44), P01637 (SEQID NO: 45), P01835 (SEQ ID NO: 46), P01836 (SEQ ID NO: 47), and P01837(SEQ ID NO: 48) identified by BLAST searches.

SEQUENCE LISTING

-   SEQ ID NO:1 Human MASP-2-   SEQ ID NO:2 Part of heavy chain of NimoAb101 including the variable    region-   SEQ ID NO:3 Part of light chain of NimoAb101 including the variable    region-   SEQ ID NO:4 Part of heavy chain of NimoAb101 including the variable    region-   SEQ ID NO:5 Part of light chain of NimoAb101 including the variable    region-   SEQ ID NO:6 CDR1 of heavy chain (also designated H1) of NimoAb101-   SEQ ID NO:7 CDR2 of heavy chain (also designated H2) of NimoAb101-   SEQ ID NO:8 CDR3 of heavy chain (also designated H3) of NimoAb101-   SEQ ID NO:9 CDR1 of light chain (also designated L1) of NimoAb101-   SEQ ID NO:10 CDR2 of light chain (also designated L2) of NimoAb101-   SEQ ID NO:11 CDR3 of light chain (also designated L3) of NimoAb101-   SEQ ID NO:12 PCR primer-   SEQ ID NO:13 PCR primer-   SEQ ID NO:14 MASP-2 (FIG. 2)-   SEQ ID NO:15 MASP-1 (FIG. 2)-   SEQ ID NO:16 C1r (FIG. 2)-   SEQ ID NO:17 C1s (FIG. 2)-   SEQ ID NO:18 Nucleotide sequence encoding the variable region of the    light chain of the NimoAb101 antibody (DWE16140-6cons, FIG. 8)-   SEQ ID NO:19 Variable region of the light chain of the NimoAb101    antibody (DWE16140-6cons, FIG. 8)-   SEQ ID NO:20 Nucleotide sequence encoding the variable region of the    heavy chain of NimoAb101 antibody (DWE16140-3consRev, FIG. 9)-   SEQ ID NO:21 Variable region of the heavy chain of NimoAb101    antibody (DWE16140-3consRev, FIG. 9)-   SEQ ID NO:22 P18525 (FIG. 10)-   SEQ ID NO:23 P18526 (FIG. 10)-   SEQ ID NO:24 P18529 (FIG. 10)-   SEQ ID NO:25 P01764 (FIG. 10)-   SEQ ID NO:26 P01783 (FIG. 10)-   SEQ ID NO:27 DWE16140-4con (FIG. 10)-   SEQ ID NO:28 DWE16140-5con (FIG. 10)-   SEQ ID NO:29 DWE16140-1con (FIG. 10)-   SEQ ID NO:30 DWE16140-2con (FIG. 10)-   SEQ ID NO:31 DWE16140-3con (FIG. 10)-   SEQ ID NO:32 DWE16140-8con (FIG. 10)-   SEQ ID NO:33 P01868 (FIG. 10)-   SEQ ID NO:34 P01869 (FIG. 10)-   SEQ ID NO:35 P20759 (FIG. 10)-   SEQ ID NO:36 P20760 (FIG. 10)-   SEQ ID NO:37 DWE16140-6con (FIG. 11)-   SEQ ID NO:38 DWE16140-7con (FIG. 11)-   SEQ ID NO:39 DWE16140-9con (FIG. 11)-   SEQ ID NO:40 DWE16140-10con (FIG. 11)-   SEQ ID NO:41 P01594 (FIG. 11)-   SEQ ID NO:42 P01595 (FIG. 11)-   SEQ ID NO:43 P01635 (FIG. 11)-   SEQ ID NO:44 P01636 (FIG. 11)-   SEQ ID NO:45 P01637 (FIG. 11)-   SEQ ID NO:46 P01835 (FIG. 11)-   SEQ ID NO:47 P01836 (FIG. 11)-   SEQ ID NO:48 P01837 (FIG. 11)

Definitions

The term “C-terminal part of MASP-2” refers to the C-terminus of MASP-2comprising the EGF, CUB2, CCP1, CCP2 and serine protease domains,wherein the C-terminal part of MASP-2 does not include the CUB1 domain.

The term “epitope” refers to a specific site on a compound, i.e. aprotein to which a certain antibody specifically binds. An epitope maybe linear, i.e. a peptide or an epitope may be a three dimensionalstructure.

DETAILED DESCRIPTION OF THE INVENTION

Antibodies

It is one aspect of the present invention to provide antibodies orfunctional equivalents thereof specifically recognising and binding anepitope within the C-terminal part of MASP-2 or a functional homologuethereof. The epitope may be any of the epitopes mentioned herein below.

The antibody or functional equivalent thereof may be any antibody knownin the art, for example a polyclonal or a monoclonal antibody derivedfrom a mammal or a synthetic antibody, such as a single chain antibodyor hybrids comprising antibody fragments. Furthermore, the antibody maybe mixtures of monoclonal antibodies or artificial polyclonalantibodies. In addition functional equivalents of antibodies may beantibody fragments, in particular epitope binding fragments.Furthermore, antibodies or functional equivalent thereof may be smallmolecule mimic, micking an antibody. Naturally occurring antibodies areimmunoglobulin molecules consisting of heavy and light chains. Inpreferred embodiments of the invention, the antibody is a monoclonalantibody.

Monoclonal antibodies (Mab's) are antibodies, wherein every antibodymolecule are similar and thus recognises the same epitope. Monoclonalantibodies are in general produced by a hybridoma cell line. Methods ofmaking monoclonal antibodies and antibody-synthesizing hybridoma cellsare well known to those skilled in the art. Antibody producinghybridomas may for example be prepared by fusion of an antibodyproducing B lymphocyte with an immortalized B-lymphocyte cell line.Monoclonal antibodies according to the present invention may for examplebe prepared as described in Antibodies: A Laboratory Manual, By EdHarlow and David Lane, Cold Spring Harbor Laboratory Press, 1988. Saidmonoclonal antibodies may be derived from any suitable mammalianspecies, however frequently the monoclonal antibodies will be rodentantibodies for example murine or rat monoclonal antibodies. It ispreferred that the antibodies according to the present invention aremonoclonal antibodies or derived from monoclonal antibodies.

Polyclonal antibodies is a mixture of antibody molecules recognising aspecific given antigen, hence polyclonal antibodies may recognisedifferent epitopes within said antigen. In general polyclonal antibodiesare purified from serum of a mammal, which previously has been immunizedwith the antigen. Polyclonal antibodies may for example be prepared byany of the methods described in Antibodies: A Laboratory Manual, By EdHarlow and David Lane, Cold Spring Harbor Laboratory Press, 1988.Polyclonal antibodies may be derived from any suitable mammalianspecies, for example from mice, rats, rabbits, donkeys, goats, sheeps,cows or camels. The antibody is preferably not derived from anon-mammalian species, i.e. the antibody is for example preferably not achicken antibody. The antibody may also for example be an artificialpolyclonal antibody as for example described in U.S. Pat. No. 5,789,208or 6,335,163, both patent specifications are hereby incorporated byreference into the application in their entirety.

In one embodiment of the invention the antibody is a human antibody,such as a human monoclonal antibody. Human antibodies may be made tohuman target molecules for example by protein engineering, by selectionfrom synthetic libraries, or by immunization of transgenic mice carryinghuman antibody genes.

Alternatively, the antibody may be a humanised antibody. Humanisedantibodies are in general chimeric antibodies comprising regions derivedfrom a human antibody and regions derived from a non-human antibody,such as a rodent antibody. Humanisation (also called Reshaping orCDR-grafting) is a well-established technique for reducing theimmunogenicity of monoclonal antibodies (mAbs) from xenogeneic sources(commonly rodent) and for improving their activation of the human immunesystem. frameworks in which to graft the rodent CDRs. The term“humanised antibody molecule” (HAM) is used herein to describe amolecule having an antigen binding site derived from an immunoglobulinfrom a non-human species, whereas some or all of the remainingimmunoglobulin-derived parts of the molecule is derived from a humanimmunoglobulin. The antigen binding site may comprise: either a completevariable domain from the non-human immunoglobulin fused onto one or morehuman constant domains; or one or more of the complementaritydetermining regions (CDRs) grafted onto appropriate human frameworkregions in the variable domain. One method for humanising MAbs relatedto production of chimeric antibodies in which an antigen binding sitecomprising the complete variable domains of one antibody are fused toconstant domains derived from a second antibody, preferably a humanantibody. Methods for carrying out such chimerisation procedures are forexample described in EP-A-0 120 694 (Celltech Limited), EP-A-0 125 023(Genentech Inc.), EP-A-0 171 496 (Res. Dev. Corp. Japan), EPA-0173494(Stanford University) and EP-A-0 194 276 (Celltech Limited). A morecomplex form of humanisation of an antibody involves the re-design ofthe variable region domain so that the amino acids constituting thenon-human antibody binding site are integrated into the framework of ahuman antibody variable region (Jones et al., 1986).

The antibodies according to the present invention may also berecombinant antibodies. Recombinant antibodies are antibodies orfragments thereof or functional equivalents thereof produced usingrecombinant technology. For example recombinant antibodies may beproduced using a synthetic library or by phage display. Recombinantantibodies may be produced according to any conventional method forexample the methods outlined in “Recombinant Antibodies”, FrankBreitling, Stefan Dübel, Jossey-Bass, September 1999.

The antibodies according to the present invention may also be bispecificantibodies, i.e. antibodies specifically recognising two differentepitopes. Bispecific antibodies may in general be prepared starting frommonoclonal antibodies, or from recombinant antibodies, for example byfusing two hybridoma's in order to combine their specificity, byChemical crosslinking or using recombinant technologies. Antibodiesaccording to the present invention may also be tri-specific antibodies.

Functional equivalents of antibodies may in one preferred embodiment bea fragment of an antibody, preferably an antigen binding fragment or avariable region. Examples of antibody fragments useful with the presentinvention include Fab, Fab′, F(ab′) 2 and Fv fragments. Papain digestionof antibodies produces two identical antigen binding fragments, calledthe Fab fragment, each with a single antigen binding site, and aresidual “Fc” fragment, so-called for its ability to crystallizereadily. Pepsin treatment yields an F(ab′)₂ fragment that has twoantigen binding fragments which are capable of crosslinking antigen, anda residual other fragment (which is termed pFc′). Additional fragmentscan include diabodies, linear antibodies, single-chain antibodymolecules, and multispecific antibodies formed from antibody fragments.As used herein, “functional fragment” with respect to antibodies, refersto Fv, F(ab) and F(ab′)₂ fragments.

Preferred antibody fragments retain some or essential all the ability ofan antibody to selectively binding with its antigen or receptor. Somepreferred fragments are defined as follows:

-   (1) Fab is the fragment that contains a monovalent antigen-binding    fragment of an antibody molecule. A Fab fragment can be produced by    digestion of whole antibody with the enzyme papain to yield an    intact light chain and a portion of one heavy chain.-   (2) Fab′ is the fragment of an antibody molecule and can be obtained    by treating whole antibody with pepsin, followed by reduction, to    yield an intact light chain and a portion of the heavy chain. Two    Fab′ fragments are obtained per antibody molecule. Fab′ fragments    differ from Fab fragments by the addition of a few residues at the    carboxyl terminus of the heavy chain CH1 domain including one or    more cysteines from the antibody hinge region.-   (3) (Fab′)₂ is the fragment of an antibody that can be obtained by    treating whole antibody with the enzyme pepsin without subsequent    reduction. F(ab′)₂ is a dimer of two Fab′ fragments held together by    two disulfide bonds.-   (4) Fv is the minimum antibody fragment that contains a complete    antigen recognition and binding site. This region consists of a    dimer of one heavy and one light chain variable domain in a tight,    non-covalent association (V_(H)-V_(L) dimer). It is in this    configuration that the three CDRs of each variable domain interact    to define an antigen binding site on the surface of the V_(H)-V_(L)    dimer. Collectively, the six CDRs confer antigen binding specificity    to the antibody. However, even a single variable domain (or half of    an Fv comprising only three CDRs specific for an antigen) has the    ability to recognize and bind antigen, although at a lower affinity    than the entire binding site.

In one embodiment of the present invention the antibody is a singlechain antibody (“SCA”), defined as a genetically engineered moleculecontaining the variable region of the light chain, the variable regionof the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule. Such single chain antibodiesare also referred to as “single-chain Fv” or “scFv” antibody fragments.Generally, the Fv polypeptide further comprises a polypeptide linkerbetween the VH and VL domains that enables the scFv to form the desiredstructure for antigen binding.

The antibody may also be selected for useful properties, for example itmay be desirable to control serum half life of the antibody. In general,complete antibody molecules have a very long serum persistence, whereasfragments (<60-80 kDa) are filtered very rapidly through the kidney.Glycosylation on complete antibodies in general, prolongs serumpersistence. Hence, if long term action of the MASP-2 antibody isdesirable, the MASP-2 antibody is preferably a complete antibody,whereas if shorter action of the MASP-2 antibody is desirable, anantibody fragment might be preferred.

In another embodiment of the present invention the functional equivalentof an antibody is a small molecule mimic, mimicking an antibody.

Preferred antibodies within the scope of the present invention areantibodies or functional equivalents thereof capable of inhibiting thefunction of MASP-2. The activity of MASP-2 may be the serine proteaseactivity of MASP-2, such as serine protease activity to C4 and/or to C2.In particular, antibodies or functional equivalents thereof capable ofinhibiting the serine protease activity of MASP-2 are preferred. Evenmore preferred are antibodies or functional equivalents thereof capableof inhibiting C4 deposition of MBL-MASP-2 complexes. Yet more preferredantibodies according to the present invention are antibodies orfunctional equivalents thereof capable of inhibiting C4 deposition infull serum. Yet more preferred antibodies or functional equivalentsthereof are capable of inhibiting C4 deposition in full serum fromindividuals with C4 disposition activity. Useful assays for determiningC4 deposition are described herein below.

In addition, preferred antibodies, are antibodies or functionalequivalents thereof capable of inhibiting C2 deposition of MBL-MASP-2complexes. Yet more preferred antibodies according to the presentinvention are antibodies or functional equivalents thereof capable ofinhibiting C2 deposition in full serum. Yet more preferred antibodies orfunctional equivalents thereof are capable of inhibiting C2 depositionin full serum from individuals with C2 disposition activity. Usefulassays for determining C2 deposition are described herein below.

Hence, preferred antibodies or functional equivalents thereof arecapable of inhibiting C4 and/or C2 deposition in full serum to less than50%, such as less than 40%, for example less than 30%, such as less than25%, for example less than 20%, such as less than 15%, for example lessthan 10%, such as less than 5% of control C4 deposition. Preferably, theantibody is capable of inhibiting C4 deposition in full serum to lessthan 30%, preferably less than 25%, more preferably less than 20%, evenmore preferably less than 15%, yet more preferably less than 10%.Alternatively or in addition, preferred antibodies are capable ofinhibiting C2 deposition in full serum to less than 30%, preferably lessthan 25%, more preferably less than 20%, even more preferably less than15%, yet more preferably less than 10%.

In one very preferred embodiment of the invention the antibody isselected from the group of monoclonal antibodies produced by thehybridoma cell lines deposited under accession number 03050904.Furthermore, the functional equivalents may be fragments, preferablybinding fragments of said antibodies.

In one embodiment of the present invention the antibody or functionalequivalent thereof comprises specific hypervariable regions, designatedCDR. Preferably, the CDRs are CDRs according to the Kabat CDRdefinition. CDRs or hypervariable regions may for example be identifiedby sequence alignment to other antibodies. Preferably, the antibody orfunctional equivalent thereof comprises at least one, more preferably atleast 2, even more preferably all three of the following heavy chainCDRs:

-   -   1. H1 of the DWE16140-4con indicated in FIG. 10 (SEQ ID 6);    -   2. H2 of the DWE16140-4con indicated in FIG. 10 (SEQ ID 7);    -   3. H3 of the DWE16140-4con indicated in FIG. 10 (SEQ ID 8)

More preferably, the antibody or functional equivalent thereof comprisesa heavy chain comprising or consisting of a sequence which is at least95%, more preferably at least 98%, even more preferably at least 99%homologous or identical to SEQ ID 4. Yet more preferably, the antibodyor functional equivalent thereof comprises a heavy chain comprising orconsisting of the sequence set forth in SEQ ID 4.

Even more preferably the antibody or functional equivalent thereofcomprises a heavy chain comprising or consisting of a sequence which isat least 95%, yet more preferably at least 98%, even more preferably atleast 99% homologous or identical to SEQ ID NO:2. Yet more preferablythe heavy chain consists of the sequence DWE16140-4 (SEQ ID NO:27) ofFIG. 10.

% homology may be determined as described herein for functionalhomologues of MASP-2. Most preferably, the antibody or functionalequivalent thereof comprises a heavy chain comprising or consisting ofthe sequence set forth in SEQ ID 2. Preferably, said antibody orfunctional equivalent thereof is capable of specifically recognising theepitope recognised by the antibody designated NimoAb101.

In another embodiment of the present invention the antibody orfunctional equivalent thereof comprises specific hypervariable regions,designated CDR. Preferably, the CDRs are CDRs according to the Kabat CDRdefinition. Preferably, the antibody or functional equivalent thereofcomprises at least one, more preferably at least 2, even more preferablyall three of the following light chain CDRs:

-   -   4. L1 of the DWE16140-10con indicated in FIG. 11 (SEQ ID 9);    -   5. L2 of the DWE16140-10con indicated in FIG. 11 (SEQ ID 10);    -   6. L3 of the DWE16140-10con indicated in FIG. 11 (SEQ ID 11)

More preferably, the antibody or functional equivalent thereof comprisesa light chain comprising or consisting of a sequence which is at least95%, more preferably at least 98%, even more preferably at least 99%homologous or identical to SEQ ID 5. Yet more preferably, the antibodyor functional equivalent thereof comprises a light chain comprising orconsisting of the sequence set forth in SEQ ID 5.

Even more preferably, the antibody or functional equivalent thereofcomprises a light chain comprising or consisting of a sequence which isat least 95%, yet more preferably at least 98%, even more preferably atleast 99% homologous or identical to SEQ ID NO:3. % homology may bedetermined as described herein for functional homologues of MASP-2. Morepreferably, the antibody or functional equivalent thereof comprises alight chain comprising or consisting of the sequence set forth in SEQ IDNO:3. Yet more preferably, the light chain consists of the sequenceDWE16140-10con (SEQ ID NO:39) of FIG. 11. Preferably, said antibody orfunctional equivalent thereof is capable of specifically recognizing theepitope recognized by the antibody designated NimoAb101.

In a preferred embodiment the antibody or functional equivalent thereofcomprises the CDRs of the heavy chain and the CDRs of the light chaindescribed herein above. More preferably, the antibody or functionalequivalent thereof comprises the variable region of the heavy chaindescribed above and the variable region of the light chain describedabove. Even more preferably, the antibody or functional equivalentthereof comprises the heavy chain described herein above and the lightchain described herein above. Thus, in a very preferred embodiment theinvention relates to an antibody comprising one or more, preferably atleast 2, even more preferably at least 3, yet more preferably at least4, even more preferably at least 5, yet more preferably all 6 CDRsselected from the group consisting of

-   -   1) CDR1 of the heavy chain of SEQ ID 6;    -   2) CDR2 of the heavy chain of SEQ ID 7;    -   3) CDR3 of the heavy chain of SEQ ID 8;    -   4) CDR1 of the light chain of SEQ ID 9;    -   5) CDR1 of the light chain of SEQ ID 10; and    -   6) CDR1 of the light chain of SEQ ID 11.        or a functional equivalent thereof. This antibody furthermore,        preferably is capable of inhibiting MASP-2 activity and/or        capable of specifically recognising a MASP-2 epitope as        described herein below.        MASP-2 Epitopes and Peptides

The MASP-2 protein comprises of a number of domains namely the CUB1,EGF, CUB2, CCP1, CCP2 and serine protease domains. A schematicpresentation of MASP2 is given in FIG. 1. Position of the individualdomains within human MASP-2 is indicated in FIG. 2. It is believed thatthe domain responsible for association with MBL is situated in theN-terminus, whereas the serine protease domain is responsible for theserine protease activity of MASP-2. Surprisingly, antibodies raised tothe C-terminus of MASP-2 are more efficient in inhibiting the activityof MASP-2 in full serum than other antibodies to MASP-2.

The antibodies and functional equivalents thereof according to thepresent invention specifically recognises an epitope within theC-terminal part of MASP-2. “Specifically recognises” means that theantibody binds to said epitope with significantly higher affinity thanto any other molecule or part thereof. Preferably, the antibody onlybinds said epitope as detected by Western blotting or ELISA. To ensurethat the antibody specifically recognises an epitope within a givenfragment of MASP-2 said fragment may be used as antigen duringgeneration of said antibody. It is preferred within the presentinvention, that the MASP-2 antigen used for immunisation is larger than18 amino acids, for example at least 20, such as at least 25, forexample at least 30 amino acids in length. By way of example, if theantibody should recognise an epitope within the CCP1, CCP2 and serineprotease domains, a peptide consisting of the CCP1, CCP2 and serineprotease domains may be used as antigen during generation of saidantibody.

Preferably, the antibody or the functional equivalent thereofspecifically recognises an epitope within the C-terminal part of MASP-2,wherein the C-terminal part comprises or even more preferably consistsof the EGF, CUB2, CCP1, CCP2 and serine protease domains. In oneembodiment of the invention, the C-terminal part of MASP-2 comprises orpreferably consists of the CUB2, CCP1, CCP2 and serine protease domains.In another embodiment of the present invention the C-terminal part ofMASP-2 comprises or preferably consists of the CCP1, CCP2 and serineprotease domains. In yet another embodiment of the invention theC-terminal part of MASP-2 comprises or consists of the CCP2 and serineprotease domains. In a preferred embodiment of the invention theC-terminal part of MASP-2 comprises or preferably consists of the serineprotease domain.

In a still further embodiment of the invention the antibody specificallyrecognises and binds an epitope within a MASP-2 fragment that comprisesor preferably consists of the CCP1 domain. In yet another embodiment ofthe invention the antibody specifically recognises and binds an epitopewithin a MASP-2 fragment that comprises or preferably consists of theCCP2 domain. In yet a further embodiment of the invention the antibodyspecifically recognises and binds an epitope within a MASP-2 fragmentthat comprises or preferably consists of the CCP1 and CCP2 domains.

By the term “MASP-2” is meant any MASP-2 molecule known to the personskilled in the art. Said MASP-2 may for example be derived from amammal, for example MASP2 may be derived from a human being. In apreferred embodiment of the present invention, MASP-2 is human MASP-2 asidentified by SEQ ID 1 or a functional homologue thereof sharing atleast 50%, preferably at least 60%, more preferably at least 70%, evenmore preferably at least 80%, yet more preferably at least 90%, yet evenmore preferably at least 95% homology or more preferably identity withSEQ ID 1. In a very preferred embodiment of the invention MASP-2 isMASP-2 of SEQ ID 1.

Hence, in a preferred embodiment the antibody specifically recognisesand binds an epitope within a MASP-2 fragment that comprises or consistsof aa 136 to 686 of SEQ ID 1 or a functional equivalent thereof, hencesaid fragment comprises the EGF, CUB2, CCP1, CCP2 and serine proteasedomains of human MASP-2.

In another embodiment of the invention the antibody specificallyrecognises and binds an epitope within a MASP-2 fragment that comprisesor consists of aa 183 to 686 of SEQ ID 1 or a functional equivalentthereof. Said fragment thus comprises the CUB2, CCP1, CCP2 and serineprotease domains of human MASP-2.

In yet another embodiment of the invention, the antibody specificallyrecognises and binds an epitope within a MASP-2 fragment that comprisesor consists of aa 293 to 362 of SEQ ID 1 or a functional equivalentthereof. Said fragment comprises the CCP1 domain of human MASP-2.

In a further embodiment of the invention the antibody specificallyrecognises and binds an epitope within a MASP-2 fragment that comprisesor consists of aa 293 to 431 of SEQ ID 1 or a functional homologuethereof. Said fragment comprises the CCP1 and CCP2 domains of humanMASP-2.

In a still further embodiment of the present invention said antibodyspecifically recognises and binds an epitope within a MASP-2 fragmentthat comprises or consists of aa 363 to 431 of SEQ ID 1 or a functionalequivalent thereof. Said fragment comprises the CCP2 domain of humanMASP-2.

In an even further embodiment of the present invention the antibodyspecifically recognises and binds an epitope within a MASP-2 fragmentthat comprises or consists of aa 293 to 686 of SEQ ID 1 or a functionalequivalent thereof. Said fragment comprises the CCP1, CCP2 and serineprotease domains of human MASP-2.

In yet a further embodiment of the present invention the antibodyspecifically recognises and binds an epitope within a MASP-2 fragmentthat comprises or consists of aa 363 to 686 of SEQ ID 1 or a functionalequivalent thereof. Said fragment(=CCP2, serine protease)

In a yet even further embodiment the antibody specifically recognisesand binds an epitope within a MASP-2 fragment that comprises or consistsof aa 445 to 686 of SEQ ID 1 or a functional equivalent thereof. Saidfragment comprises the serine protease domain of human MASP-2.

In one embodiment of the present invention the epitope is not within aa505 to 523 and aa 538 to 556 of SEQ ID 1.

In one preferred embodiment of the invention the antibody specificallyrecognises and binds an epitope within a MASP-2 fragment that comprisesor preferably consists of aa 363 to 385, such a 370 to 390, for example380 to 400, such a 390 to 410, for example 400 to 420, such a 410 to430, for example 420 to 440, such a 430 to 450, for example 440 to 460,such a 450 to 470, for example 460 to 480, such a 470 to 490, forexample 480 to 500, such a 490 to 510, for example 500 to 520, such a510 to 530, for example 520 to 540, such a 530 to 550, for example 540to 560, such a 550 to 570 for example 560 to 580, such a 570 to 590, forexample 580 to 600, such a 590 to 610, for example 600 to 620, such a610 to 630, for example 620 to 640, such a 630 to 650, for example 640to 660, such a 650 to 670, for example 660 to 686 of SEQ ID 1, whereinsaid fragment at the most comprises 100, preferably at the most 80, morepreferably at the most 60, even more preferably at the most 40 aminoacids.

In another preferred embodiment, the antibody specifically recognisesand binds an epitope within a MASP-2 fragment that comprises orpreferably consists of aa 400 to 420, such a 410 to 430, for example 420to 440, such a 430 to 450, for example 440 to 460, such a 450 to 470,for example 460 to 480, such a 470 to 490, for example 480 to 500, sucha 490 to 510, for example 500 to 520, such a 510 to 530, for example 520to 540, such a 530 to 550 of SEQ ID 1, wherein said fragment at the mostcomprises 100, preferably at the most 80, more preferably at the most60, even more preferably at the most 40 amino acids.

In yet another preferred embodiment the antibody specifically recognisesand binds an epitope within a MASP-2 fragment that comprises orpreferably consists of aa 410 to 430, for example 420 to 440, such a 430to 450, for example 440 to 460 of SEQ ID 1, wherein said fragment at themost comprises 100, preferably at the most 80, more preferably at themost 60, even more preferably at the most 40 amino acids.

In another very preferred embodiment the antibody specificallyrecognises and binds an epitope within a MASP-2 fragment that comprisesor preferably consists of aa 420 to 440 or aa 430 to 450.

In one preferred embodiment of the present invention the antibodies orfunctional equivalents thereof specifically recognises the epitoperecognised by the monoclonal antibody produced by the hybridoma cellline designated M0545YM035.

In another preferred embodiment of the present invention the antibodiesor functional equivalents thereof specifically recognises the epitoperecognised by the monoclonal antibody produced by the hybridoma cellline designated M0545YM029.

In another preferred embodiment of the present invention the antibodiesor functional equivalents thereof specifically recognises the epitoperecognised by the monoclonal antibody produced by the hybridoma cellline designated M0545YM046.

In another preferred embodiment of the present invention the antibodiesor functional equivalents thereof specifically recognises the epitoperecognised by the monoclonal antibody produced by the hybridoma cellline designated M0545YM048.

In one especially preferred embodiment of the present invention theantibodies or functional equivalents thereof specifically recognises theepitope recognised by the monoclonal antibody produced by the hybridomacell line deposited under the deposition number 03050904.

In particular, the antibodies produced by the hybridoma cell linedeposited under the deposition number 03050904 and the hybridoma celllines designated M0545YM029 and M0545YM035 recognise overlappingepitopes. Thus it is preferred that the antibodies or functionalequivalents thereof specifically recognises an epitope or part thereofrecognised by one or more selected from the group consisting of:

-   -   the monoclonal antibody produced by the hybridoma cell line        deposited under the deposition number 03050904;    -   the hybridoma cell line designated M0545YM029; and    -   the hybridoma cell line designated M0545YM035

According to the present invention, when a given antibody recognises atleast part of an epitope recognised by another given antibody, these twoantibody are said to recognise the same or overlapping epitopes.

Different assays available to the person skilled in the art may be usedto determine whether an antibody (also designated test antibody)recognises the same or an overlapping epitope as a particular monoclonalantibody (also designated reference antibody). Preferably, the assayinvolves the steps of:

-   -   Providing MASP-2 or a fragment thereof comprising the epitope        recognised by the reference antibody    -   Add the test antibody and the reference antibody to the said        MASP-2, wherein either the test antibody or the reference        antibody is labelled with a detectable label. Alternatively,        both antibodies may be labelled with different detectable labels    -   Detecting the presence of the detectable label at MASP-2    -   Thereby detecting whether the test antibody may displace the        reference antibody

If the reference antibody is displaced, the test antibody recognises thesame or an overlapping epitope as the reference antibody. Thus if thereference antibody is labelled with a detectable label, then a lowdetectable signal at MASP-2 is indicative of displacement of thereference antibody. If the test antibody is labelled with a detectablelabel, then a high detectable signal at MASP-2 is indicative ofdisplacement of the reference antibody. The MASP-2 fragment maypreferably be immobilised on a solid support enabling facile handling.The detectable label may be any directly or indirectly detectable label,such as an enzyme, a radioactive isotope, a heavy metal, a colouredcompound or a fluorescent compound. In example 5 in the section “MASP-2competitive ELISA” herein below one very preferred method of determiningwhether a test antibody recognises the same or an overlapping epitope asa reference antibody is described. The person skilled in the art mayeasily adapt said method to the particular antibodies in question.

It is also an object of the present invention to provide isolated MASP-2polypeptides useful as antigens for generation of MASP-2 antibodies, inparticular MASP-2 antibodies capable of inhibiting the activity ofMASP-2 in full serum. Said polypeptides may for example be used toimmunise an animal in order to generate antibodies to the polypeptides.

Any C-terminal MASP-2 polypeptide may be used with the presentinvention, preferred polypeptides are however isolated polypeptidescomprising or more preferably consisting of the EGF, CUB2, CCP1, CCP2and serine protease domains of MASP-2. Hence, a very preferred MASP-2polypeptide according to the invention comprises or even more preferablyconsists of aa 136 to 686 of SEQ ID 1 or a functional equivalentthereof.

In another embodiment, the isolated MASP-2 polypeptide comprises orpreferably consists of the CUB2, CCP1, CCP2 and serine protease domains.Hence, a preferred MASP-2 polypeptide comprises or consists of aa 183 to686 of SEQ ID 1 or a functional equivalent thereof.

In yet another embodiment of the present invention the isolated MASP-2polypeptide comprises or preferably consists of the CCP1 domain. Hence,a preferred polypeptide comprises or even more preferably consists of aa293 to 362 of SEQ ID 1 or a functional equivalent thereof.

In a further embodiment of the invention the isolated MASP-2 polypeptidecomprises or preferably consists of the CCP2 domain. For example, thepolypeptide may comprise or more preferably consist of aa 363 to 431 ofSEQ ID 1 or a functional equivalent thereof.

In a yet further embodiment of the invention the isolated MASP-2polypeptide comprises or preferably consists of the CCP1 and CCP2domains. Hence, the polypeptide may comprises or even consist of aa 293to 431 of SEQ ID 1 or a functional equivalent thereof.

The C-terminal MASP-2 polypeptides are preferably at the most 570 aminoacids long, for example the polypeptides may be in the range of 20 to570 amino acids, such as 30 to 500, for example 50 to 400, such as 100to 300, for example 150 to 250 amino acids long.

Functional equivalents or functional homologues of MASP-2 polypeptidesor fragments comprising a predetermined amino acid sequence, for examplea fragment of the amino acid sequence outlined in SEQ ID Tare defined aspolypeptides comprising an amino acid sequence capable of beingrecognised by an antibody also capable of recognising the predeterminedamino acid sequence. The terms “functional equivalent” and “functionalhomologue” are used interchangeably herein.

Functional homologues according to the present invention comprisepolypeptides with an amino acid sequence, which are sharing a homologywith the predetermined MASP2 polypeptide sequences as outlined hereinabove. For example such polypeptides are at least about 40 percent, suchas at least about 50 percent homologous, for example at least about 60percent homologous, such as at least about 70 percent homologous, forexample at least about 75 percent homologous, such as at least about 80percent homologous, for example at least about 85 percent homologous,such as at least about 90 percent homologous, for example at least 92percent homologous, such as at least 94 percent homologous, for exampleat least 95 percent homologous, such as at least 96 percent homologous,for example at least 97 percent homologous, such as at least 98 percenthomologous, for example at least 99 percent homologous with thepredetermined polypeptide sequences as outlined herein above.

Homology may preferably be calculated by any suitable algorithm or bycomputerised implementations of such algorithms for example CLUSTAL inthe PC/Gene program by Intelligenetics or GAP, BESTFIT, BLAST, FASTA andTFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup (GCG). The homology between amino acid sequences may furthermorebe calculated with the aid of well known matrices such as for exampleany one of BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55, BLOSUM60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85,and BLOSUM 90.

Functional homologues according to the present invention are preferablypolypeptides with an amino acid sequence, which is at least about 50percent, preferably at least about 60 percent, more preferably at leastabout 70 percent, even more preferably at least about 75 percent, yetmore preferably at least about 80 percent, even more preferably at leastabout 85 percent, yet more preferably at least about 90 percent, evenmore preferably at least 95 percent homologous, most preferably at least98 percent identical with the predetermined MASP-2 polypeptide sequencesas outlined herein above.

Functional homologues may comprise an amino acid sequence that comprisesat least one substitution of one amino acid for any other amino acid.For example such a substitution may be a conservative amino acidsubstitution or it may be a non-conservative substitution. Preferably,said substitutions are conservative substitution.

A conservative amino acid substitution is a substitution of one aminoacid within a predetermined group of amino acids for another amino acidwithin the same group, wherein the amino acids within a predeterminedgroups exhibit similar or substantially similar characteristics. Withinthe meaning of the term “conservative amino acid substitution” asapplied herein, one amino acid may be substituted for another withingroups of amino acids characterised by having

-   i) polar side chains (Asp, Glu, Lys, Arg, His, Asn, Gln, Ser, Thr,    Tyr, and Cys,)-   ii) non-polar side chains (Gly, Ala, Val, Leu, Ile, Phe, Trp, Pro,    and Met)-   iii) aliphatic side chains (Gly, Ala Val, Leu, Ile)-   iv) cyclic side chains (Phe, Tyr, Trp, His, Pro)-   v) aromatic side chains (Phe, Tyr, Trp)-   vi) acidic side chains (Asp, Glu)-   vii) basic side chains (Lys, Arg, His)-   viii) amide side chains (Asn, Gln)-   ix) hydroxy side chains (Ser, Thr)-   x) sulphor-containing side chains (Cys, Met), and-   xi) amino acids being monoamino-dicarboxylic acids or    monoaminomonocarboxylic-monoamidocarboxylic acids (Asp, Glu, Asn,    Gln).

The addition or deletion of an amino acid may be an addition or deletionof from 2 to 5 amino acids, such as from 5 to 10 amino acids, forexample from 10 to 20 amino acids, such as from 20 to 50 amino acids.However, additions or deletions of more than 50 amino acids, such asadditions from 50 to 200 amino acids, are also comprised within thepresent invention.

In addition to the polypeptide compounds described herein, stericallysimilar compounds may be formulated to mimic the key portions of thepeptide structure and that such compounds may also be used in the samemanner as the peptides of the invention. This may be achieved bytechniques of modelling and chemical designing known to those of skillin the art. For example, esterification and other alkylations may beemployed to modify the amino terminus of, e.g., a di-arginine peptidebackbone, to mimic a tetra peptide structure. It will be understood thatall such sterically similar constructs fall within the scope of thepresent invention.

Peptides with N-terminal alkylations and C-terminal esterifications arealso encompassed within the present invention. Functional equivalentsalso comprise glycosylated and covalent or aggregative conjugates,including dimers or unrelated chemical moieties. Such functionalequivalents are prepared by linkage of functionalities to groups whichare found in fragment including at any one or both of the N- andC-termini, by means known in the art.

Functional equivalents may thus comprise fragments conjugated toaliphatic or acyl esters or amides of the carboxyl terminus, alkylaminesor residues containing carboxyl side chains, e.g., conjugates toalkylamines at aspartic acid residues; O-acyl derivatives of hydroxylgroup-containing residues and N-acyl derivatives of the amino terminalamino acid or amino-group containing residues, e.g. conjugates withMet-Leu-Phe. Derivatives of the acyl groups are selected from the groupof alkyl-moieties (including C3 to C10 normal alkyl), thereby formingalkanoyl species, and carbocyclic or heterocyclic compounds, therebyforming aroyl species. The reactive groups preferably are difunctionalcompounds known per se for use in cross-linking proteins to insolublematrices through reactive side groups.

Functional homologues may furthermore be polypeptide encoded by anucleic acid which is able to hybridise to the complementary strand of anucleic acid sequence encoding the predetermined MASP-2 polypeptidesequences as outlined herein above under stringent conditions.

Stringent conditions as used herein shall denote stringency as normallyapplied in connection with Southern blotting and hybridisation asdescribed e.g. by Southern E. M., 1975, J. Mol. Biol. 98:503-517. Forsuch purposes it is routine practise to include steps ofprehybridization and hybridization. Such steps are normally performedusing solutions containing 6×SSPE, 5% Denhardt's, 0.5% SDS, 50%formamide, 100 μg/ml denatured salmon testis DNA (incubation for 18 hrsat 42° C.), followed by washings with 2×SSC and 0.5% SDS (at roomtemperature and at 37° C.), and a washing with 0.1×SSC and 0.5% SDS(incubation at 68° C. for 30 min), as described by Sambrook et al.,1989, in “Molecular Cloning/A Laboratory Manual”, Cold Spring Harbor),which is incorporated herein by reference.

The epitope(s) recognised by a specific antibody may be determined byany conventional method, for example methods involving the use of massspectrometry. Non-limiting examples of methods of epitope mapping usingmass spectrometry include:

-   1. Baerga-Ortiz, A, Hughes, C A, Mandell, J G, Komives, E A: Epitope    mapping of a monoclonal antibody against human thrombin by    H/D-exchange mass spectrometry reveals selection of a diverse    sequence in a highly conserved protein. Protein Sci. 11:1300-1308,    2002-   2. Hochleitner, E O, Borchers, C, Parker, C, Bienstock, R J, Tomer,    K B: Characterization of a discontinuous epitope of the human    immunodeficiency virus (HIV) core protein p24 by epitope excision    and differential chemical modification followed by mass    spectrometric peptide mapping analysis.-   3. Hochleitner, E O, Gorny, M K, Zolla-Pazner, S, Tomer, K B: Mass    spectrometric characterization of a discontinuous epitope of the HIV    envelope protein HIV-gp120 recognized by the human monoclonal    antibody 1331A. J. Immunol. 164:4156-4161, 2000-   4. Parker, C E, Tomer, K B: MALDI/MS-based epitope mapping of    antigens bound to immobilized antibodies. Mol. Biotechnol. 20:49-62,    2002-   5. Peter, J F, Tomer, K B: A general strategy for epitope mapping by    direct MALDI-TOF mass spectrometry using secondary antibodies and    cross-linking. Anal. Chem. 73:4012-4019, 2001-   6. Van De, W J, Deininger, S O, Macht, M, Przybylski, M, Gershwin, M    E: Detection of molecular determinants and epitope mapping using    MALDI-TOF mass spectrometry. Clin. Immunol. Immunopathol.    85:229-235, 1997-   7. Yu, L, Gaskell, S J, Brookman, J L: Epitope mapping of monoclonal    antibodies by mass spectrometry: identification of protein antigens    in complex biological systems. J. Am. Soc. Mass Spectrom. 9:208-215,    1998-   8. Zhao, Y, Chalt, B T: Protein epitope mapping by mass    spectrometry. Anal. Chem. 66:3723-3726, 1994    Methods of Preparing MASP-2 Antibodies

The antibodies and functional equivalents thereof may be produced by anysuitable method known to the person skilled in the art.

One method of producing an antibody specifically recognising and bindingan epitope within the C-terminal part of MASP-2 comprises the step ofadministering to a mammal the C-terminal part of MASP-2 or a fragmentthereof or a functional homologue thereof. Said C-terminal part ofMASP-2 or a fragment thereof or a functional homologue thereof may beany of the MASP-2 fragments and peptides described herein above. Inparticular, the MASP-2 fragment may be any of the MASP-2 fragmentsdescribed herein above, wherein said fragments comprise an epitope. TheC-terminal part of MASP-2 or fragment thereof or functional homologuethereof administrated to said mammal is also designated the “MASP-2antigen” herein.

In one embodiment, the present invention relates to methods of producingan antibody capable of inhibiting the activity of MASP-2, wherein saidantibody specifically recognises an epitope within the C-terminal partof MASP-2.

The MASP-2 antigen is preferably at least 18 amino acids in length, morepreferably at least 20, even more preferably at least 25 amino acids inlength.

The MASP-2 antigen may be administrated to said mammal more than once,such as twice, for example 3 times, such as 3 to 5 times, for example 5to 10 times, such as 10 to 20 times, for example 20 to 50 times, such asmore than 50 times. It is also possible that different MASP-2 antigensare administered to the same mammal, either simultaneously ofsequentially in any order.

In general, the MASP-2 antigen will be in an aqueous solution orsuspension prior to administration. Furthermore, the MASP-2 antigen maybe mixed with one or more other compounds. For example, the MASP-2antigen may be mixed with one or more suitable adjuvants and/or with oneor more carriers.

Adjuvants are any substance whose admixture with an administered antigenincreases or otherwise modifies the immune response to said antigen.Adjuvants may for example be selected from the group consisting ofAlK(SO₄)₂, AlNa(SO₄)₂, AlNH₄ (SO₄), silica, alum, Al(OH)₃, Ca₃(PO₄)₂,kaolin, carbon, aluminum hydroxide, muramyl dipeptides,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP),N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred toas nor-MDP),N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′2′-dipalmitoyl-sn-glycero-3-hydroxphosphoryloxy)ethylamine(CGP 19835A, also referred to as MTP-PE), RIBI (MPL+TDM+CWS) in a 2%squalene/Tween-80® emulsion, lipopolysaccharides and its variousderivatives, including lipid A, Freund's Complete Adjuvant (FCA),Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides (forexample, poly IC and poly AU acids), wax D from Mycobacterium,tuberculosis, substances found in Corynebacterium parvum, Bordetellapertussis, and members of the genus Brucella, liposomes or other lipidemulsions, Titermax, ISCOMS, Quil A, ALUN (see U.S. Pat. Nos. 58,767 and5,554,372), Lipid A derivatives, choleratoxin derivatives, HSPderivatives, LPS derivatives, synthetic peptide matrixes or GMDP,Interleukin 1, Interleukin 2, Montanide ISA-51 and QS-21. Preferredadjuvants to be used with the invention include Freund's CompleteAdjuvant (FCA), Freund's Incomplete Adjuvants.

Carriers are scaffold structures, e.g. a polypeptide or apolysaccharide, to which an antigen is capable of being associated. Acarrier may be present independently of an adjuvant. The function of acarrier can for example be to increase the molecular weight of inparticular MASP-2 antigen in order to increase the immunogenicity, toconfer stability, to increase the biological activity, or to increaseserum half-life. The carrier may be any suitable carrier known to theperson skilled in the art, for example a protein or an antigenpresenting cell. A carrier protein could be, but is not limited tokeyhole limpet hemocyanin, serum proteins such as transferrin, bovineserum albumin, human serum albumin, thyroglobulin or ovalbumin,immunoglobulins, or hormones, such as insulin or palmitic acid.

The MASP-2 antigen may be administered by any suitable method, forexample parenterally, orally or topically. Preferably, however theantigen is administered by injection, for example intramuscular,intradermal, intravenous or subcutaneous injection, more preferably bysubcutaneous or intravenous injection.

The mammal may be any suitable mammal. Monoclonal antibodies arefrequently prepared using a rodent, for example a mouse or a ratPolyclonal antibodies may be prepared by administering the MASP-2antigen to any mammal, for example mice, rats, rabbits, donkeys, goats,sheeps, cows or camels. Antibodies according to the invention may alsobe mixtures of antibodies, such as mixtures of monoclonal antibodies,mixtures of polyclonal antibodies or both. Hence, it is also comprisedwithin the invention that more than one kind of animal may be used.

If the antibody is a monoclonal antibody, antibody producing cells areusually isolated from said mammal subsequent to immunisation. The methodmay for example comprise the steps of isolating antibody producing cellsfrom said mammal, preparing hybridoma cells from said antibody producingcells, cultivating said hybridomas and isolating antibodies produced bysaid hybridomas.

For example said cells may be isolated from said mammal 1 day, such asin the range of 2 to 10 days, for example in the range of 10 to 20 days,such as in the range of 20 to 40 days, for example in the range of 1 to3 months, such as in the range of 3 to 6 months, for example in therange of 6 to 12 months, such as in the range of 12 to 24 months, forexample more than 24 months after first administration of the MASP-2antigen.

The antibody producing cells are in general B-cells and said cells mayfor example be isolated from said mammal by excising the spleen of saidmammal.

Once the antibody producing cells have been isolated from said mammal,the cells may be fused with other cells in order to obtain hybridomacells. Said cells may for example be cancer cells, such as cells derivedfrom a leukaemia, for example myeloma cells. After fusion said hybridomacells may be cultivated using standard cultivation protocols. Thecultivation medium (supernatant) may be tested for the presence ofsuitable MASP2 antibodies and hybridoma cells capable of producingsuitable MASP-2 antibodies may be selected and cultivated.

Testing may be performed by any suitable method, for example methodsdetecting the presence of antibodies capable of associating with theMASP-2 antigen. Such methods include, but are not limited to Westernblotting, ELISA (Enzyme-Linked Immunosorbent Assay), dot-blotting orTRIFMA. In addition or alternatively, said cultivation medium may betested for the presence of MASP-2 antibodies capable of inhibitingMASP-2 activity. Suitable assays to determine MASP-2 activity aredescribed herein below.

Once hybridoma cells capable of producing suitable MASP-2 antibodieshave been identified, said cells may be cultivated using any standardprotocol and antibodies produced by said cells may be purified.Purification of antibodies may be done using any standard protocol, forexample purification using anti-Ig antibodies, protein G or protein A.

If the antibody is a polyclonal antibody, said antibody may for examplebe purified directly from serum from a mammal, immunised with the MASP-2antigen. Purification may be done using any standard method, for examplepurification using anti-Ig antibodies, protein G or protein A.

Methods of preparing monoclonal antibodies, mixtures of monoclonalantibodies or polycloncal antibodies are for example described inAntibodies: A Laboratory Manual, By Ed Harlow and David Lane, ColdSpring Harbor Laboratory Press, 1988.

One non-limiting example of a method to prepare antibodies according tothe present invention is described in example 1 herein below.

Depending of the nature of the antibody, several other methods may beemployed.

In one embodiment of the invention the antibody may be produced usingrecombinant methods, for example protein engineering or by screening oflibraries. Libraries may be synthetic libraries or libraries comprisingnatural material. One useful method is phage display. In general phagedisplay, involves screening one or more phage libraries for phagesencoding a useful antibody or functional equivalent thereof.

In another embodiment of the present invention, the methods involve useof animals, for example rodents, such as mice genetically engineered toproduce chimeric antibodies or antibodies of another species, forexample human antibodies. For example, transgenic animals, such astransgenic mice, carrying antibody genes from another species, such ashuman antibody genes, may be immunised with any of the above mentionedMASP-2 fragments.

Antibody fragments may be produced by fragmentation of the antibodiesaccording to the invention using any method known to the person skilledin the art. The methods include, but are not limited to digestion withone or more proteases, for example papain or pepsin, as well asreduction or a combination of both.

Inhibiting MASP-2 Activity

The present invention also relates to methods of inhibiting the activityof MASP-2. In particular, the methods may involve the steps of

-   -   1) Providing a composition comprising MASP-2;    -   2) Providing a MASP-2 antibody according to the invention;    -   3) Incubating said composition with said antibody, thereby        inhibiting MASP-2 activity

The composition may be any composition comprising MASP-2, for exampleserum. The MASP-2 antibody is preferably a MASP-2 antibody capable ofinhibiting the activity of MASP-2.

Assays to Detect MASP-2 Activity

MASP-2 activity may be determined by any suitable assay. Useful assaysinclude the in particular assays, wherein serine protease activity ofMASP-2/MBL complexes are tested. Preferred assays, are assaysdetermining inhibition of C2 and/or C4 deposition.

The assays may involved the steps of preparing a solid surface on whichan MBL associating agent is immobilised, binding MBL/MASP-2 complexes tosaid MBL associating agent and screening for inhibition of MASP-2catalysed reactions.

The solid surface may be any useful solid surface, for examplemicrotiter wells. The MBL associating agent, may be any compound towhich MBL binds with high affinity, for example MBL antibodies, mannanor mannose, preferably however it is mannan. The MBL/MASP-2 complexesmay be derived from any suitable source, it may for example berecombinant MBL, recombinant MASP-2 or MBL and/or MASP-2 purified fromserum. Recombinant MBL/MASP-2 may be full length MBL/MASP-2 orfunctional fragments thereof. Furthermore, recombinant MBL/MASP-2 may beattached to one or more other compounds, such as genetic tags. MBLand/or MASP-2 may be derived from any suitable species for example itmay be human MBL/MASP-2. In one embodiment of the invention, theMBL/MASP-2 complexes are found in full serum and are not purified priorto performing the assay. Said assays then test inhibition of depositionof substrate, i.e. C4 in full serum. The MASP-2 catalysed reaction ispreferably deposition of C2 and/or C4.

The antibody or functional equivalent thereof to be screened forinhibition activity is added to the bound MBL/MASP-2. The antibody mayhave been purified or it may be for example crude hybridoma cell culturesupernatant. Controls without added antibody are preferably alsoperformed. The antibody may be added in concentrations in the range of 1μg/ml to 500 μg/ml, preferably in the range of 5 μg/ml to 400 μg/ml,more preferably in the range of 10

1 μg/ml to 300 μg/ml, even more preferably in the range of 15 μg/ml to200 μg/ml, yet more preferably in the range of 20 to 100 μg/ml.

A MASP-2 substrate is added to the MBL/MASP-2 complexes. Preferably,said substrate is either C2 or C4 or a mixture of both. The substratemay be recombinantly produced or a serum derived substrate. Thesubstrate may or may not have been purified prior to use, but preferablyit is purified. In order to monitor deposition, the substrate, may belabeled with a detectable label, for example with an enzyme, aradioactive compound, a fluorescent compound, a dye, a heavy metal, achemilumniscent compound or the like.

It is however preferred that deposition is detected using specificbinding agent, such as an antibody, specifically recognising digestedsubstrate. For example, antibodies recognising human complement C4c maybe used. Said antibodies may be labelled, by a directly or indirectlydetectable label. For example by an enzyme, a radioactive compound, afluorescent compound, a dye, a heavy metal, a chemilumniscent compoundor an affinity compound. Affinity compounds includes for example otherantibodies or biotin, streptavidin.

The above mentioned steps may be performed in any useful order, i.e.substrate may be added before or simultaneously to inhibiting antibody,MBL/MASP-2 complexes may be mixed with the substrate and/or theinhibiting antibody prior to immobilisation on a solid surface etc. Thesteps may also be performed in the order described.

If MBL/MASP-2 complexes, substrate and antibody are mixed prior toimmobilisation, then said mixture may be preincubated for a given time,for example preincubation may be in the range of 5 min to 2 hours. Ingeneral, MBL/MASP-2, substrate and antibody is premixed, when MBL/MASP-2complexes are present in serum and have not previously been purifiedfrom serum.

In one preferred embodiment of the present invention, the activity ofMASP-2 is determined using any of the methods described in examples 2and 3. In particular, antibodies capable of inhibiting C4 deposition,should preferably be able to inhibit C4 deposition in at least one,preferably both of the methods described in example 2 and 3. Antibodiescapable of inhibiting C4 deposition, should more preferably at least beable to inhibit C4 deposition according to the methods described inexample 2, whereas antibodies capable of inhibiting C4 deposition infull serum should be capable of inhibiting C4 deposition in full serumas described in example 3.

Pharmaceutical Compositions and Administration Thereof

In one embodiment the present invention relates to pharmaceuticalcompositions comprising the antibodies and functional equivalentsthereof according to the invention. The invention furthermore relates tomedicaments for treatment of a clinical condition comprising theantibody, methods of treatment of a clinical condition comprisingadministration of said antibody or use of said antibody for preparationof a medicament for treatment of a clinical condition.

The clinical condition may be any of the conditions mentioned hereinbelow. The individual in need of administration of MASP-2 antibodies maybe any individual suffering from said condition or at risk of acquiringsaid clinical condition. Preferably, the individual is a human being.

Treatment may be curative, palliative, ameliorating and/or prophylactictreatment.

The pharmaceutical compositions of the present invention preferablycomprise a pharmaceutical effective amount of at least one antibody orfunctional equivalent thereof specifically recognising an epitope withinthe C-terminus of MASP-2 (herein above and below designated “MASP-2antibody”). A pharmaceutical effective amount is an amount of MASP-2antibody, which in induces the desired response in an individualreceiving said pharmaceutical composition.

The pharmaceutically effective amount of the MASP-2 antibody depends onthe individual to which it should be administered, in particular on thesize of said individual as well as the clinical condition and thespecific mode of administration. In general however, in the range of 1mg to 5000 mg, preferably in the range of 10 mg to 3000 mg, morepreferably in the range of 50 mg to 1000 mg, for example in the range of100 mg to 750 mg, such as in the range of 150 mg to 500 mg, for examplein the range of 200 mg to 400 mg, such as in the range of 250 mg to 350mg, for example around 300 mg MASP-2 antibody should be administered toan adult human being per dose.

The composition of the present invention may be a pharmaceuticalcomposition suitable for parenteral administration. Such compositionspreferably, include aqueous and non-aqueous sterile injection solutionswhich may contain wetting or emulsifying reagents, anti-oxidants, pHbuffering agents, bacteriostatic compounds and solutes which render theformulation isotonic with the body fluid, preferably the blood, of theindividual; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents or thickening agents. The pharmaceuticalcomposition may be presented in unit-dose or multi-dose containers, forexample, sealed ampoules and vials and may be stored in a freeze-driedcondition requiring only the addition of the sterile liquid carrierimmediately prior to use.

Preferably, the composition of the present invention comprises one ormore suitable pharmaceutical excipients, which could be non-sterile orsterile, for use with cells, tissues or organisms, such as apharmaceutical excipients suitable for administration to an individual.Such excipients may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations of theseexcipients in various amounts. The formulation should suit the mode ofadministration. The invention further relates to pharmaceutical kit ofparts comprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.Examples of non-aqueous excipients are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate.

Preferably, the pharmaceutical compositions of the present invention areprepared in a form which is injectable, either as liquid solutions orsuspensions; furthermore solid forms suitable for solution in orsuspension in liquid prior to injection are also within the scope of thepresent invention. The preparation may be emulsified or the immunogenicdeterminant as well as the collectins and/or collectin homologuesaccording to the present invention may be encapsulated in liposomes.

The MASP-2 antibody may be administered alone or in combination withother compounds, either simultaneously or sequentially in any order.

Administration could for example be parenteral injection or infusion,rapid infusion, nasopharyngeal absorption, dermal absorption, andenterally, such as oral administration. Parenteral injection could forexample be intravenous, intramuscular, intradermal or subcutaneousinjection. Preferably, said administration is parenterally by injectionor infusion.

The MASP-2 antibody should be administered as often as required, hencethe MASP-2 antibody may be administered more than once, such as at leasttwo times, for example at least 3 times, such as at least 4 times, forexample at least 5 times, such as in the range of 1 to 100 times, forexample in the range of 1 to 50 times, such as in the range of 1 to 25times, for example in the range of 1 to 10 times.

Preferably, there is at least 1 day between 2 administrations, such asat least 2 days, for example at least 3 days, such as at least 5 days,for example at least one week, such as at least 2 weeks, for example atleast one month, such as at least 6 months, for example at least 1 year,such at least 2 years, for example at least 3 years, such as at least 5years, for example at least 10 years.

Clinical Conditions

The clinical condition according to the present invention may be anycondition, which may be treated curative, ameliorating or prophylacticby administration of MASP-2 antibodies.

The clinical condition may be one preferred embodiment of the presentinvention be a chronic inflammatory disease. Chronic inflammatorydiseases may for example be autoimmune inflammatory conditions.

Autoimmune inflammatory conditions (also designated “autoimmunedisorders” herein) may be loosely grouped into those primarilyrestricted to specific organs or tissues and those that affect theentire body. Examples of organ-specific disorders (with the organaffected) include multiple sclerosis (myelin coating on nerveprocesses), type I diabetes mellitus (pancreas), Hashimotos thyroiditis(thyroid gland), pernicious anemia (stomach), Addison's disease (adrenalglands), myasthenia gravis (acetylcholine receptors at neuromuscularjunction), rheumatoid arthritis (joint lining), uveitis (eye), psoriasis(skin), Guillain-Barre Syndrome (nerve cells) and Grave's disease(thyroid). Systemic autoimmune diseases include systemic lupuserythematosus, glomeronephritis and dermatomyositis.

In one embodiment of the present invention the preferred clinicalcondition is selected from the group consisting of rheumatoid arthritisand systemic lupus erythermatosis.

Other examples of autoimmune disorders include asthma, eczema, atopicaldermatitis, contact dermatitis, other eczematous dermatitides,seborrheic dermatitis, rhinitis, Lichen planus, Pemplugus, bullousPemphigoid, Epidermolysis bullosa, uritcaris, angioedemas, vasculitides,erythemas, cutaneous eosinophilias, Alopecia areata, atherosclerosis,primary biliary cirrhosis and nephrotic syndrome. Related diseasesinclude intestinal inflammations, such as Coeliac disease, proctitis,eosinophilia gastroenteritis, mastocytosis, inflammatory bowel disease,Chrohn's disease and ulcerative colitis, as well as food-relatedallergies.

In another embodiment of the present invention the clinical condition ischaracterised by massive cell loss, for example due to apoptosis ornecrosis. Said necrosis or apoptosis may be induced by a number ofdifferent factors.

In a preferred embodiment of the invention, the clinical condition isischemia/reperfusion injury. Ischemia may arise from various differentcauses, for example after stroke, myocardial infarction, major surgeryor organ transplantation. Thus, the clinical condition may beischemia/reperfusion injury caused by for example stroke, myocardialinfarction, major surgery or organ transplantation.

Hence, the clinical condition may be ischemia/reperfusion injury, whichis a result of PTCA (percutaneous transluminal coronary angioplasty) orCABG (coronary artery bypass grafting). Furthermore, the clinicalcondition may be ischemia/reperfusion injury, which is a result of acutemyocardial infarction or brain ischemia.

EXAMPLES Example 1

Monoclonal Anti-MASP-2 Antibody:

Female Wistar rats (8 weeks old) were injected subcutaneous with 3 μgrecombinant human MASP-2 CCP1-CCP2-serine protease domain (CCP1/2-SP)(Rossi et al., 2001) emulsified in complete Freund's adjuvant andboosted three times with the 3 μg CCP1/2-SP in incomplete Freund'sadjuvant. Three days prior to removal of the spleen the rat was boostedintravenously with 3 μg CCP1/2-SP in saline.

Fusion of a suspension of spleen cells with mouse myeloma cells(X63-Ag8.653) and culture on mouse feeder cells were as described (Liuet al., 2001).

For the detection of anti-MASP-2 antibody in the supernatants,microtiter plates (FluoroNunc, Nunc, Kamstrup, Denmark) were coated with1 μg mannan (Nakajima and Ballou, 1974) in 100 μl 15 mM Na₂CO₃, 35 mMNaHCO₃, 1.5 mM NaN₃, pH 9.6 (coating buffer) overnight at 4° C. Residualprotein-binding sites were blocked by 200 μg human serum albumin (HSA)in 200 μl of 10 mM Tris-HCl, 140 mM NaCl, 1.5 mM NaN₃, pH 7.4 (TBS) for1 hour at room temperature.

The wells were washed in TBS containing 0.05% (v/v) Tween 20 and 5 mMCaCl₂) (TBS/Tw/Ca²⁺) followed by incubation overnight at 4° C. with 0.5μg MBL/MASP preparation in 100 μl TBS/Tw/Ca²⁺.

After wash, hybridoma supernatants diluted 5 fold in TBS/Tw/Ca²⁺ wereadded to the wells and incubated for 2 hours at room temperature. Boundanti-MASP-2 antibodies were detected by adding 50 ng rabbit-anti rat Igantibody (Dako, Glostrup, Denmark) labelled with europium (Perkin Elmer,Gaithersburg, USA) (Hemmila et al., 1984) in 100 μl TBS/Tw, 25 μM EDTA.After 1 hour at room temperature the wells were washed and boundeuropium was detected by the addition of 200 μl enhancement solution(Perkin Elmer), and reading the time resolved fluorescence on a DELFIA®fluorometer (Perkin Elmer). Selected positive cultures were cloned twiceby limiting dilution and frozen in 60% (v/v) DMEM, 30% (v/v) fetal calfserum, 10% (v/v) DMSO.

An MBL/MASP preparation was separated on SDS-PAGE followed by blottingonto a PVDF membrane. The selected antibodies were tested forrecognition of MASP-2 on the blot.

Inhibiting antibodies were identified by screening for inhibition ofMASP-2 catalysed C4 deposition as described in example 2 herein below.

Culture supernatant of hybridoma cell lines producing the selectedantibodies was centrifuged at 10,000 g for 15 minutes and thesupernatant buffed with 10 mM Na₂HPO₄, 10 mM EDTA, pH 7. One hundred mlsupernatant was passed through a 1 ml anti-bovine Ig column (5 mganti-bovine Ig (Dako) per ml CNBr activated Sepharose 4B beads (AmershamBioscience, Uppsala, Sweden)) equilibrated in 1.5 mM KH₂PO₄, 8.1 mMNa₂HPO₄, 137 mM NaCl, 2.7 mM KCl, pH 7.4 (PBS) with 10 mM EDTA(PBS/EDTA). The effluent was passed onto a 1 ml protein G Sepharose(Amersham Bioscience) column pre-washed with 0.1 M glycine, pH 2.5 andre-equilibrated with PBS/EDTA. The columns were washed with 30 mlPBS/EDTA and eluted with 0.1 M glycine pH 2.5. The eluate was collectedin fractions of 0.5 ml into 40 μl 1 M Tris-HCL, pH 8.5.

Example 2

Assay for Inhibition of MASP-2 Catalysed C4 Deposition:

The assay is composed of three steps 1) preparation of mannan coatedmicrotiter wells 2) binding of rMBL and rMASP-2 to mannan coated wells3) screening for inhibition of MASP-2 catalysed C4 deposition.

1) Preparation of Mannan Coated Microtiter Wells:

96 wells microtiter plates (FluroNunc, Nalgene Nunc Int., Denmark) arecoated with mannan (10 mg/L, Sigma Chemical Co., St. Louis, USA) in acoating buffer (Na₂CO₃: 3.18 g/L; NaHCO₃: 5.86 g/L; pH adjusted to 9.6using HCl) over night at 4° C. Wells are washed twice in TBS (10 mMTris, 150 mM NaCl, pH adjusted to 7.4 using HCl). Wells are then blockedby incubation for 1 hr at room temperature in a buffer as above exceptthat 1 mg/mL of human albumin is added (State Serum Institute,Copenhagen Denmark). Wells are washed 3 times in TBST+Ca²⁺ (10 mM Tris,150 mM NaCl, 10 mM CaCl₂); 0.05% Tween 20, pH adjusted to 7.4 using HCl,from now on washing buffer) and are now ready for use.

2) Binding of rMBL and rMASP-2 to Mannan Coated Wells:

0.8 ng/well of recombinant purified human His-tagged MASP-2 and 1ng/well of recombinant purified human MBL are bound to mannan coatedmicrotiter wells by incubation over night at 4° C. in the above washingbuffer except that 1 mg/mL of human albumin is added (State SerumInstitute, Copenhagen Denmark). Wells are then washed 3 timers inwashing buffer and are ready for use.

3) Screening for Inhibition of MASP-2 Catalysed C4 Deposition:

The substance to be screened for inhibition activity (e.g. hybridomacell culture supernatant, purified antibody) is added to rMBL/rMASP-2bound to mannan coated microtiter wells in the above washing bufferexcept that 1 mg/mL of human albumin is added (State Serum Institute,Copenhagen Denmark) and incubated for 1 hr at room temperature. Wellsare washed 3 times in washing buffer and incubated 1.5 hr at 37° C. withpurified human complement component C4 (approx. 1.5-2 ng/mL) in a bufferof barbital sodium (5 mM), NaCl (181 mM), CaCl2) (2.5 mM), MgCl2 (1.25mM), pH 7.4, 1 mg/mL of human albumin (State Serum Institute, CopenhagenDenmark) is added before use. Wells are washed 3 times in washing bufferand 0.89 mg/L biotinylated rabbit anti-human complement component C4c isadded (Dako, Denmark, biotinylated according to standard procedures).Wells are incubated for 1 hr at room temperature and washed 3 times inwashing buffer. Europium labelled streptavidin (Wallac, Turku, Finland)is added at a concentration of 0.1 mg/L in the above washing bufferexcept that calcium is omitted and 50 μM EDTA is included. Wells areincubated 1 hr at room temperature and washed 3 times in washing buffer.Wells are developed by adding 100 μL of Delfia Enhancement Solution(Perkin Elmer Wallac, Norton, USA) and incubated on an orbital shakerfor 5 min. at room temperature. Wells are counted in a Wallac Victor 2dMulti counter 1420 (Wallac, Turku, Finland).

Inhibition is seen as decreased counts compared to wells where noinhibiting substance has been added.

Example 3

Assay for Inhibition of MASP-2 Catalysed C4 Deposition in Full Serum:

The serum sample to be analysed is diluted 250 times (finalconcentration) in the above barbital buffer and C4 is added (1.5-2ng/mL, final concentration). The substance to be screened for inhibitionactivity (e.g. purified antibody) is added and samples are incubatedbetween 5 min and 2 hours at 37° C. Normally incubation is 15 min. 100p1is added to mannan coated microtiter wells prepared as described aboveand incubated 1% hr at 37° C. Wells are washed 3 times in the abovewashing buffer and 0.89 mg/L biotinylated rabbit anti-human complementcomponent C4c is added (Dako, Denmark, biotinylated according tostandard procedures). Wells are incubated for 1 hr at room temperatureand washed 3 times in washing buffer. Europium labelled streptavidin(Wallac, Turku, Finland) is added at a concentration of 0.1 mg/L in theabove washing buffer except that calcium is omitted and 50 μM EDTA isincluded. Wells are incubated 1 hr at room temperature and washed 3times in washing buffer. Wells are developed by adding 100 μL of DelfiaEnhancement Solution (Perkin Elmer Wallac, Norton, USA) and incubated onan orbital shaker for 5 min. at room temperature. Wells are counted in aWallac Victor 2d Multi counter 1420 (Wallac, Turku, Finland).

FIG. 3 illustrates the Eu3 counts obtained after performing theinhibition assay using either the monoclonal antibody produced by thehybridoma cell line deposited under 03050904, PBS or mannose. Theantibody is capable of largely inhibiting C4 deposition in all theserums tested.

FIG. 4 illustrates inhibition of C4 deposition in full serum by themonoclonal antibody produced by the hybridoma cell line deposited under03050904 as a function of concentration of the antibody.

Example 4

Pharmaceutical Composition

One dosis unit of a pharmaceutical composition according to theinvention may comprise 300 mg MASP-2 antibody produced by the hybridomacell line deposited under deposition number 03050904, purified asdescribed in example 1, and formulated in 10 mM Tris-buffer, pH 7.4 and140 mM NaCl.

The pharmaceutical composition is filtered through a Planova 75N and aPlanova 35N filter to remove any vira, and sterile filtered over a 0.22μm filter.

This formulation is suitable for parenteral administration to a humanadult.

Example 5

Production and Characterisation of Inhibitory Anti-huMASP-2 MurineMonoclonal Antibodies

Immunization

Four mice (6-8 weeks old) were immunized. A total of four injections wasadministered (50 μg antigen per animal per injection). Mice wereinjected on day 0 with equal parts (v/v) of complete Freund's adjuvantand 10 μg of His-tagged human MASP2 (N039-76C). Three following boostswere carried out with equal parts (v/v) incomplete Freund's adjuvant andantigen, with three weeks between boosts. The best responding mouse 10days after the last injection was selected using an ELISA test againstMASP2.

Hybridization, Fusion

Fusions were performed using conventional methodology. The spleniclymphocytes of the best responding animal were fused with the cell lineSp2/O—Ag-14 using PEG (polyethylene glycol), and the resultinghybridomas were seeded in 96-well plates in HAT medium.

Medium was changed 2-3 times before screening. Usually, hybridomacolonies were ready for screening in 3-5 weeks. Supernatants were testedfor the presence of inhibitory antibody by C4 deposition assay. Thehybridomas were sub-cloned by limiting dilution.

785 primary clones were screened for inhibition activity and 50inhibiting clones were selected for subcloning. Subclones were screenedfor inhibition and clones showing the most inhibitory activity weresubcloned again. After 2 to 3 subclonings, 4 inhibitory clones ofinterest remained (see table 1 herein below).

Inhibition of C-4 Deposition Assay

Buffer B1/HSA: a buffer of barbital sodium (5 mM), NaCl (181 mM), CaCl₂)(2.5 mM), MgCl2 (1.25 mM), pH 7.4. 1 mg/mL of human albumin (State SerumInstitute, Copenhagen Denmark) is added before use.

The assay is composed of three steps 1) preparation of mannan coatedmicrotiter wells 2) Preincubation of antibodies with human serum 3)measurement of MASP-2 catalysed C4 deposition.

Preparation of Mannan Coated Microtiter Wells:

96 wells microtiter plates (FluroNunc, Nalgene Nunc Int., Denmark) arecoated with mannan (10 mg/L, Sigma Chemical Co., St. Louis, USA) in acoating buffer (Na2CO3: 3.18 g/L; NaHCO3: 5.86 g/L; pH adjusted to 9.6using HCl) over night at room temperature. Wells are washed 2 times inTBS (10 mM Tris, 150 mM NaCl, pH=7.4 using HCl). Wells are then blockedby incubation for 1 hr at room temperature in a buffer as above exceptthat 1 mg/mL of human albumin is added (State Serum Institute,Copenhagen Denmark). Wells are then washed 3 times in TBST (10 mM Tris,150 mM NaCl, 0.05% Tween 20, pH=7.4 using HCl, from) and are ready foruse.

Preincubation of Antibodies with Human Serum:

The antibodies to be tested are serial diluted in B1/HSA. 100 μL fromeach dilution is transferred to a well of a 96-well Nucleon surfaceplate. 100 μL full human serum diluted×125 (final 250×) is added to eachwell together with purified human complement component C4 (approx. 3-4mg/L). Incubate at 37 C 15 min. 100 μL of the antibody-human serum mixis then transferred to the previous made mannan coated plates.

Measurement of MASP-2 Catalysed C4 Deposition:

The mannan coated plates are then incubated 1.5 hr at 37° C. Wells arewashed 3 times in washing buffer TBST+Ca(10 mM Tris, 150 mM NaCl, 10 mMCaCl₂), 0.05% Tween 20, pH=7.4 using HCl) and 0.89 mg/L biotinylatedrabbit anti-human complement component C4c diluted in TBST+Ca is added(Dako, Denmark, biotinylated according to standard procedures). Wellsare incubated for 1 hr at room temperature and washed 3 times in washingbuffer. Europium labelled streptavidin (Wallac, Turku, Finland) is addedat a concentration of 0.1 mg/L in the above washing buffer except thatcalcium is omitted and 50 μM EDTA is included. Wells are incubated 1 hrat room temperature and washed 3 times in washing buffer. Wells aredeveloped by adding 100 μL of Delfia Enhancement Solution (Perkin ElmerWallac, Norton, USA) and incubated on an orbital shaker for 5 min. atroom temperature. Wells are counted in a Wallac Victor 2d Multi counter1420 (Wallac, Turku, Finland).

Western Blot

Human serum (0.15 μl/lane) was run on NuPage 4-12% Bis-Trispolyacrylamide gels and transferred to PVDF membrane. The blots wereblocked with 0.5% gelatine in incubation buffer (5×: 250 mM Tris; 750 mMNaCl; 25 mM EDTA; 0.5% IGEPAL CA-630. pH 7.4) and incubated with themonoclonal antibodies, followed by HRP-conjugated rabbit anti-mouse IgG(DAKO Po260). The blots were detected with SuperSignal West PicoChemiluminescent kit (Pierce Inc.)

MASP-2 Competitive ELISA

Coating buffer: PBS (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mMNa2HPO4 pH=7.2 using NaOH)

Blocking Buffer: TBS (10 mM Tris, 150 mM NaCl, pH=7.4 using HCl)containing 1 mg/ml HSA

Wash Buffer: TBST+Ca (10 mM Tris, 150 mM NaCl, 10 mM CaCl₂), 0.05% Tween20, pH=7.4 using HCl)

Coating of ELISA Plate:

MASP-2 antigen is diluted to 1 μg/ml in coating buffer [PBS pH 7.2]. 100μl of MASP-2 coating solution is added to each well of the microtiterplate. The plate is incubated at room temperature 1 hour. The microtiterplate is emptied and each well of the plate is filled with BlockingBuffer and incubated for 1 hour at room temperature. The microtiterplate is emptied and each well is filled with Wash Buffer. Themicrotiter plate is emptied. This is repeated three times. The wells arefilled with wash buffer and stored at 4° C.

The murine antibodies are appropriately diluted (0.2 and 1.0 ug/mlfinal) with Wash Buffer Biotinylated NimoAb101 is diluted (0.2 ug/mlfinal) in wash Buffer. The diluted murine Antibodies are added (100ul/well) to the MASP-2 coated plate. The plate is incubated for 1 hourat room temperature. The microtiter plate is emptied.

The microtiter plate is washed by completely filling the wells with WashBuffer and emptying. This step is repeated twice for a total of threewashes.

Europium labelled streptavidin (Wallac, Turku, Finland) is added at aconcentration of 0.1 mg/L in the above washing buffer except thatcalcium is omitted and 50 μM EDTA is included. Wells are incubated 1 hrat room temperature. The microtiter plate is emptied and the wells arewashed three times with Wash Buffer. Wells are developed by adding 100μL of Delfia Enhancement Solution (Perkin Elmer Wallac, Norton, USA) andincubated on an orbital shaker for 5 min. at room temperature. Wells arecounted in a Wallac Victor 2d Multi counter 1420 (Wallac, Turku,Finland).

Results

Table 1 indicates four hybridomas producing inhibitory antibodiesidentified as outlined above.

TABLE 1 NimoAb- trival Name hybridoma-ID name NimoAb104 M0545YM035 035NimoAb108 M0545YM029 029 NimoAb109 M0545YM046 046 NimoAb110 M0545YM048048Potency of the New Antibodies

The four described hybridoma clones were transformed to serum freegrowth and antibodies were purified from culture supernatant by MEPHyperCel purification. The ability to inhibit lectin pathway in a fullhuman serum was determined by the C4-deposition assay as describedabove. The results are shown in FIG. 5.

From the data it can be concluded that the antibody (035) is at least3.9 times more potent than NimoAb101 in inhibition of MASP-2 activity,whereas the antibody (029) is at least 30 times more potent. The controlantibody (002) is a non-inhibitory antibody obtained during thescreening that most probably binds to the N-terminus of MASP-2. Theantibodies (046) and (048) also inhibit, but they are less potent thanNimoAb101.

Epitope Mapping

Western Blot:

Westerns were made in order to distinguish between binding of theantibodies to the N terminal A-chain (dimerization and MBL-binding part)or to the C-terminal B-chain (serine protease part of MASP-2). Humanserum was run on a reduced SDS-PAGE and immunodetection was done withthe four antibodies separate. Full length nonactivated MASP-2 willexhibit a band of 74 kDa, the A-chain and the B-chain exhibit bands of47 kDa and 27 kDa respectively. FIG. 6 shows the results of the Westernblots against MASP-2 in human serum using the different murineantibodies.

It can be concluded from the blot that all four antibodies recognize a27 kDa band (B-chain), whereas the A-chain (47 kDa) not could bedetected. Thus all four antibodies recognize a linear epitope on theB-chain.

Competitive ELISA

With the aim of elucidating whether the antibodies share the sameepitope as the rat antibody NimoAb101 we have conducted competitiveELISA. The ELISA was directed toward recombinant His-tagged MASP-2 usinga biotinylated NimoAb101 competing against two different concentrations(0.2 and 1.0 μg/ml) of the mouse antibodies.

The results are displayed in FIG. 7. The better an antibody competeswith NimoAb101, the lower the EU3+ signal will be. It can thus beconcluded that the antibodies 029(NimoAb108) and 035(NimoAb104) competevery well with the NimoAb101 antibody, thus they must share at leastpart of the epitope.

Example 6

Identification and Cloning of the VH and the VL Region of the AntibodyNimoAb101 Expressed in Hybridoma Cells Deposited Under ECACC AccessionNo. 03050904.

The specificity of antibodies resides in the complementarity determiningregions (CDRs) within the variable domains of the heavy- and lightchains. In order to characterize the monoclonal antibody Nimoab101 theVH and VL domains variable region were cloned.

Abbreviations Used

V_(H)=the variable region of the heavy chain of Ig; V_(L)=the variableregion of the light chain of Ig

CDR=complementarity-determining region; CDR1, CDR2 and CDR3=threeregions on either V_(H) or V_(L), which are numbered from the aminoterminus;

FR=framework region; FR3=the third framework region on either V_(H) orV_(L), numbered from the amino terminus

scFv=single-chain Fv fragment;

cDNA (complementary DNA)=A single-stranded DNA molecule that iscomplementary in base sequence to an RNA strand.

5″RACE=rapid amplification of cDNA 5′ end

Materials and Methods

Isolation of Total RNA and mRNA

Hybridoma cells of the cell line deposited under the ECAAC number03050904 were dispensed into 4 tubes. Cells were pelleted and thesupernatant was removed.

Two of the tubes were frozen at −80 C

Two of the tubes were used for purification of RNA using GenElute™Mammalian Total RNA Kit RTN70. RNA concentration was determinedspectrophotometrically.

Total RNA was also purified using Total RNA purification withNucleoSpin® RNA II Kit: Cat. No. 740955.20 Macherey-Nagel. The yield wasdetermined spectrophotometrically.

Poly(A) RNA was isolated from total RNA with NucleoTrap® mRNA Kit: Cat.No. 740655 Macherey-Nagel

Purified mRNA was eluted in H2O (RNase-free) and was immediately storedat −80° C.

Construction of cDNA

5′RACE

5′ rapid amplification of cDNA ends (RACE) was carried out using theSMART RACE cDNA amplification kit (Clontech) according to themanufacturer's instructions. G_(RT) was used for reverse transcription(RT), and amplification was performed with G_(3′).

The gene specific primers used in this approach were:

Oligo name Oligo sequence RACKFOR CTCATTCCTGTTGAAGCTCTTGACGA (SEQ ID 12)RACERAG1 AGGCTTGCAATCACCTCCACA (SEQ ID 13)Plasmid Construction: Cloning of PCR FragmentsParental Plasmid:

-   -   pCR2.1-TOPO (InVitrogen TOPO TA clonings kit)

The purified PCR fragment were cloned into the vector using the Toporeaction.

Sequence Analysis

DNA from plasmid mini-preparation of four recombinant clones of VL andplasmid mini-preparation of six recombinant clones of VH were sequencedof the insert in both directions using standard sequencing primers M13Fand M13R. The two respective sequences from each plasmid were assembledto a contig using VNTI contig express. The reliable part of the contigconsensus sequences were imported to NTI DNA database files.

The relevant IgG orfs were identified and translated.

Sequence Alignment and Blast Search

Computer Analysis

Sequence analysis was done with VectorNTI from Informax Inc. using theContig module.

BLAST searches were performed using NCBI home page(http://www.ncbi.nlm.nih.gov) and The European Bioinformatics Institute(EBI) (http://www.ebi.ac.uk/Tools/homology.html).

Protein alignments: All alignments were done with the VectorNTI packagefrom Informax Inc. using the ALIGN module. Multiple alignments for theIgG VL and VH proteins were done and edited with CLUSTALW and GeneDoc.

Results and Discussion

The antibody Nimoab101 was raised against the CCP1-CCP2-SP-subunit ofhuman MASP-2. We amplified the genes of the Fab fragment by PCR ofhybridoma mRNA, using primers hybridizing in the constant domains C_(H)1and C_(L) adapted from the literature (1) and primers hybridizing to anadapter that was incorporated into the the 5′ end of the cDNA genes. Thepurified VL and VH PCR products were cloned into pCR2.1-TOPO vector andsequenced with M13F and M13R primers. All of the VH sequences and all ofthe VL sequences were identical. The sequences are shown in FIGS. 8 to10.

Kabat CRD definition Light CDR1: residues 24-34 Chain CDR2: residues50-56 CDR3: residues 89-97 Heavy CDR1: residues 31-35 Chain CDR2:residues 50-65 CDR3: residues 95-102

Comparison of the sequences with published antibody variable region dataindicated that the NimoAb101 VL gene contained a leader sequence of 57nucleotides encoding leader peptide of 19 amino acid residues, while theNimoAb101 VH gene had a 60 bases long leader sequences that encoded a 20residues leader peptide.

While the light chain is a typical member of (rat-mouse-human) kappachain subgroup, the heavy chain differs from heavy chain other in thedata base. It has an insertion of 6 amino acids in CDR H3 (after residue100, Kabat numbering).

REFERENCES

-   1) Protein Sequence and Structure Analysis of Antibody Domains    published in the Spinger Verlag Laboratory Manual on Antibody    Engineering edited by Stefan Duebel and Roland Kontermann    Biological Deposition

The following biological material has been deposited at a depositaryinstitution recognised under the Budapest Treaty.

A hybridoma cell line capable of producing antibodies to MASP-2, capableof inhibiting MASP-2 activity has been deposited under the depositaccession number 03050904 with the EUROPEAN COLLECTION OF CELL CULTURES(ECACC), Salisbury, Wiltshire SP4 0JG, United Kingdom. The cell line isa hybridoma cell line derived from a fusion of rat spleen cells andmouse myeloma cells. This hybridoma cell line is producing a monoclonalantibody, which is herein referred to as NimoAb101 or NimoAb101N128-71B. The cells were deposited 9 May 2003.

A hybridoma cell line designated M0545YM035S2 (herein also designatedM0545YM035) has been deposited with Deutsche Sammlung vonMikroorganismen and Zellkulturen (DSMZ), Mascheroder Weg 1b, D-38124Braunschweig, Germany under deposit accession number DSMACC2660. Thecell line is a hybridoma cell line derived from a fusion of murinespleen cells and mouse myeloma cells. The identification reference isN162-91A-01 to 12. This hybridoma cell line is producing a monoclonalantibody, which is herein referred to as NimoAb104 or “035” or Ab035.The cells were deposited 6 May 2004.

A hybridoma cell line designated M0545YM029S2 (herein also designatedM0545YM029) has been deposited with Deutsche Sammlung vonMikroorganismen und Zellkulturen (DSMZ), Mascheroder Weg 1b, D-38124Braunschweig, Germany_under deposit accession number DSMACC2657. Thecell line is a hybridoma cell line derived from a fusion of murinespleen cells and mouse myeloma cells. The identification reference isN162-90C-01 to 12. This hybridoma cell line is producing a monoclonalantibody, which is herein referred to as NimoAb108 or “029” or Ab029.The cells were deposited 6 May 2004.

A hybridoma cell line designated M0545YM046S2 (herein also designatedM0545YM046) has been deposited with Deutsche Sammlung vonMikroorganismen und Zellkulturen (DSMZ), Mascheroder Weg 1b, D-38124Braunschweig, Germany under deposit accession number DSMACC2658. Thecell line is a hybridoma cell line derived from a fusion of murinespleen cells and mouse myeloma cells. The identification reference isN162-90D-01 to 12. This hybridoma cell line is producing a monoclonalantibody, which is herein referred to as NimoAb109 or “046” or Ab046.The cells were deposited 6 May 2004.

A hybridoma cell line designated M0545YM048S2 (herein also designatedM0545YM048) has been deposited with Deutsche Sammlung vonMikroorganismen und Zellkulturen (DSMZ), Mascheroder Weg 1b, D-38124Braunschweig, Germany under deposit accession number DSMACC2659. Thecell line is a hybridoma cell line derived from a fusion of murinespleen cells and mouse myeloma cells. The identification reference isN162-90E-01 to 12. This hybridoma cell line is producing a monoclonalantibody, which is herein referred to as NimoAb110 or “048” or Ab048.The cells were deposited 6 May 2004.

REFERENCES

-   Hemmila, I., Dakubu, S., Mukkala, V. M., Siitari, H. and    Lovgren, T. (1984) Europium as a label in time-resolved    immunofluorometric assays. Anal Biochem 137, 335-43.-   Jones, P. T., Dear, P. H., Foote, J., Neuberger, M. S., Winter,    G., 1986. Replacing the complementarity-determining regions in a    human antibody with those from a mouse. Nature, 321, 522-525-   Liu, H., Jensen, L., Hansen, S., Petersen, S. V., Takahashi, K.,    Ezekowitz, A. B., Hansen, F. D., Jensenius, J. C. and    Thiel, S. (2001) Characterization and quantification of mouse    mannan-binding lectins (MBL-A and MBL-C) and study of acute phase    responses. Scand J Immunol 53, 489-97.-   Nakajima, T. and Ballou, C. E. (1974) Characterization of the    carbohydrate fragments obtained from Saccharomyces cerevisiae mannan    by alkaline degradation. J Biol Chem 249, 7679-84.-   Rossi, V., Cseh, S., Bally, I., Thielens, N. M., Jensenius, J. C.    and Arlaud, G. J. (2001) Substrate specificities of recombinant    mannan-binding lectin-associated serine proteases-1 and -2. J Biol    Chem 276, 40880-7.

For all deposited microbial organisms mentioned in the present patentapplication the following applies.

Europe

In respect to those designations in which a European Patent is sought asample of the deposited

microorganism will be made available until the publication of themention of the grant of the European patent or until the date on whichapplication has been refused or withdrawn or is deemed to be withdrawn,only by the issue of such a sample to an expert nominated by the personrequesting the sample, and approved either i) by the Applicant and/orii) by the European Patent Office, whichever applies. (Rule 28 (4) EPC).Canada

The applicant requests that, until either a Canadian patent has beenissued on the basis of an application or the application has beenrefused, or is abandoned and no longer subject to reinstatement, or iswithdrawn, the Commissioner of Patents only authorizes the furnishing ofa sample of the deposited biological material referred to in theapplication to an independent expert nominated by the Commissioner, theapplicant must, by a written statement, inform the International Bureauaccordingly before completion of technical preparations for publicationof the international application.

Norway

The applicant hereby requests that, until the application has been laidopen to public inspection (by the Norwegian Patent Office), or has beenfinally decided upon by the Norwegian Patent Office without having beenlaid open inspection, the furnishing of a sample shall only be effectedto an expert in the art. The request to this effect shall be filed bythe applicant with the Norwegian Patent Office not later than at thetime when the application is made available to the public under Sections22 and 33(3) of the Norwegian Patents Act. If such a request has beenfiled by the applicant, any request made by a third party for thefurnishing of a sample shall indicate the ‘expert to be used. Thatexpert may be any person entered on the list of recognized experts drawnup by the Norwegian Patent Office or any person approved by theapplicant in the individual case.

Australia

The applicant hereby gives notice that the furnishing of a sample of amicroorganism shall only be effected prior to the grant of a patent, orprior to the lapsing, refusal or withdrawal of the application, to aperson who is a skilled addressee without an interest in the invention(Regulation 3.25(3) of the Australian Patents Regulations).

Finland

The applicant hereby requests that, until the application has been laidopen to public inspection (by the National Board of Patents andRegulations), or has been finally decided upon by the National Board ofPatents and Registration without having been laid open to publicinspection, the furnishing of a sample shall only be effected to anexpert in the art.

United Kingdom

The applicant hereby requests that the furnishing of a sample of amicroorganism shall only be made available to an expert. The request tothis effect must be filed by the applicant with the International Bureaubefore the completion of the technical preparations for theinternational publication of the application.

Denmark

The applicant hereby requests that, until the application has been laidopen to public inspection (by the Danish Patent Office), or has beenfinally decided upon by the Danish Patent office without having beenlaid open to public inspection, the furnishing of a sample shall only beeffected to an expert in the art. The request to this effect shall befiled by the applicant with the Danish Patent Office not later that atthe time when the application is made available to the public underSections 22 and 33(3) of the Danish Patents Act. If such a request hasbeen filed by the applicant, any request made by a third party for thefurnishing of a sample shall indicate the expert to be used. That expertmay be any person entered on a list of recognized experts drawn up bythe Danish Patent Office or any person by the applicant in theindividual case.

Sweden

The applicant hereby requests that, until the application has been laidopen to public inspection (by the Swedish Patent Office), or has beenfinally decided upon by the Swedish Patent Office without having beenlaid open to public inspection, the furnishing of a sample shall only beeffected to an expert in the art. The request to this effect shall befiled by the applicant with the International Bureau before theexpiration of 16 months from the priority date (preferably on the FormPCT/RO/I34 reproduced in annex Z of Volume I of the PCT Applicant'sGuide). If such a request has been filed by the applicant any requestmade by a third party for the furnishing of a sample shall indicate theexpert to be used. That expert may be any person entered on a list ofrecognized experts drawn up by the Swedish Patent Office or any personapproved by a applicant in the individual case.

Netherlands

The applicant hereby requests that until the date of a grant of aNetherlands patent or until the date on which the application is refusedor withdrawn or lapsed, the microorganism shall be made available asprovided in the 3 IF(1) of the Patent Rules only by the issue of asample to an expert. The request to this effect must be furnished by theapplicant with the Netherlands Industrial Property Office before thedate on which the application is made available to the public underSection 22C or Section 25 of the Patents Act of the Kingdom of theNetherlands, whichever of the two dates occurs earlier.

The invention claimed is:
 1. A method of treating a subject sufferingfrom, or at risk for developing, an ischemia/reperfusion injuryresulting from an acute myocardial infarction comprising administeringto said subject a therapeutically effective dosage of a monoclonalantibody or antigen-binding fragment thereof specifically recognizingand binding an epitope within the C-terminal part of MASP-2 consistingof the CCP1, CCP2 and serine protease domains (amino acid residues 293to 686 of SEQ ID NO:1), wherein the monoclonal antibody, orantigen-binding fragment thereof, is capable of inhibiting MASP-2catalyzed C4 deposition.
 2. The method of claim 1, wherein said antibodyis a human antibody.
 3. The method of claim 1, wherein said antibody hasbeen humanized.
 4. The method of claim 1, wherein said antigen-bindingfragment is selected from the group consisting of Fab, Fab′, F(ab′)2 andFv fragments.
 5. The method of claim 1, wherein said antibody is asingle chain antibody.